Mass spectrometer, system comprising the same, and methods for determining isotopic anatomy of compounds

ABSTRACT

A first mass spectrometer includes a first introduction device configured to select between a reference material and a first portion of an analyte and introduce the selected one of the reference material or the first portion of the analyte to an ion source, the first mass spectrometer being configured to provide third molecular analyte ions to a detector at a first mass resolution of about 30,000 or greater. A system includes the first mass spectrometer and a second mass spectrometer. A method for determining the isotopic composition of an analyte in a sample includes converting a first portion of the analyte to first molecular analyte ions, filtering out second molecular analyte ions, filtering out third molecular analyte ions, detecting two or more of the third molecular analyte ions at a mass resolution of about 30,000 or greater to determine the isotopic composition of at least a portion of the analyte.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This non-provisional application claims priority to and the benefit ofU.S. Provisional Application Ser. No. 61/712,022, filed on Oct. 10,2012, entitled SYSTEM OF MASS SPECTROMETERS FOR ISOTOPIC ANALYSIS OFINTACT, HIGH MOLECULAR WEIGHT MOLECULES, and U.S. ProvisionalApplication Ser. No. 61/869,461, filed on Aug. 23, 2013, entitledMETHODS AND APPARATUS FOR ANALYZING ISOTOPIC COMPOSITION OF MOLECULES,the entire contents of each of which are incorporated herein byreference.

FIELD

The following description generally relates to apparatus, systems andmethods for determining the isotopic anatomy of an analyte, such asmolecular gases and volatile organic compounds. More particularly, thefollowing description relates to apparatus for measuring intensityratios of molecular ions, fragment ions and adduct ions, systemsincluding the apparatus, and to methods for determining abundance ratiosof isotopologues and position-specific isotopic compositions within asample or samples.

BACKGROUND

Conventional mass spectrometry primarily focuses on measuring theconcentrations of isotopic species including only one rare isotope.These mass spectrometric techniques generally determine the overallconcentration of an isotope, irrespective of its location in themolecule (i.e., the atomic site or sites of isotopic substitution) orthe proportions of multiple isotopic substitutions within the samemolecule. Consequently, conventional mass spectrometry fails todistinguish among different isotopologues of the same molecule and thusdisregards a large amount of useful information that can be determinedfrom a complete analysis of all the different isotopologues present in asample. However, determining the isotopic composition of a moleculeincluding more than one rare isotope can provide useful information,such as the geographic origin of the molecule, temperature of origin ofthe molecule (or a sample including the molecule) or the identity of aparent molecule from which the molecule was derived.

The shortcomings of conventional mass spectrometry are particularlynoteworthy for organic compounds, which may have large numbers ofisotopologues. For example, methane (CH₄) has 57 distinct isotopicversions including various non-equivalent combinations of ¹²C, ¹³C, ¹⁴C,hydrogen, deuterium, and tritium. The number of isotopologues oflow-symmetry molecular structures grows approximately geometrically withthe number of atomic positions, meaning alkanes, lipids, sugars andother complex hydrocarbons containing several or more carbon atomstypically have at least several hundred distinct isotopologues; manysuch molecules have 10⁶ or more distinct isotopologues. Abundances ofonly a small subset of these species (typically 2-5) are meaningfullyconstrained by commonly recognized methods of isotopic analysis.

Although other methods have been developed to expand the set ofisotopologues that can be analyzed with useful precision, these methodsare applicable to a relatively narrow range of sample types and sizesand to a restricted range of isotopic species in a given analyte target.For example, demonstrated site-specific natural isotopefractionation-nuclear magnetic resonance (SNIF-NMR) techniques candetermine, for example, relative deuterium concentration and specificdeuterium-site locations in a molecule based on the deuterium NMR signalobtained for the molecule. Comparison of the relative deuteriumconcentration of the molecule with known global distributions ofhydrogen isotope concentrations can provide information regarding thegeographic origin of a sample from which the molecule was obtained.SNIF-NMR techniques, however, are not capable of analyzing abundances ofmolecules containing two or more rare isotopes at their naturalabundances and, more generally, require sample sizes that areprohibitively large for many applications and require relatively long,costly analyses. Similarly, established “clumped isotope” massspectrometric methods can analyze only a few isotopologues of small,simple, highly volatile molecules, principally because of theirinability to resolve isobaric interferences and the poor sensitivity ofexisting gas source multi-collector sector mass spectrometers. Clumpedisotope geochemistry and related techniques are described in more detailin “‘Clumped-isotope’ geochemistry—The study of naturally-occurring,multiply-substituted isotopologues,” Earth and Planetary ScienceLetters, Vol. 262, Issues 3-4, pages 309-327, the entire contents ofwhich are herein incorporated by reference.

SUMMARY

Aspects of embodiments of the invention are directed to apparatus,systems and methods for the quantitative analysis of the isotopologuesof gaseous compounds and/or volatile organic compounds. According to oneembodiment, the gaseous compounds and/or volatile organic compounds areintroduced into a gas source isotope ratio mass spectrometer, whichconverts the compounds into molecular ion, fragment ion and/or adduction beams, which are analyzed to determine the isotopic composition ofthe gaseous compounds and/or volatile organic compounds. Aspects ofembodiments are also directed to methods of data processing andstandardization for converting measured intensity ratios of isotopicspecies into abundance ratios of isotopologues, includingmultiply-substituted isotopologues and position-specific isotopiccompositions.

Embodiments of the invention are also directed to various applicationsof the apparatus, systems and methods, such as applications in earth andenvironmental science (e.g., thermometry of natural compounds anddeveloping budgets for atmospheric gases), chemistry, forensics (e.g.,chemical forensics and explosives fingerprinting), biomedical research,diagnosis and treatment of diseases (e.g., drug and/or drug metabolitetracking), and hydrocarbon (e.g., oil and gas) exploration. For example,embodiments of the invention are directed to identifying the location ofoil and gas deposits (e.g., a potential oil-field) based on the relativeproportion of isotopologues (e.g., isotopologues of methane) of a sampleas determined using the apparatus and methods described herein.

For example, one embodiment of the invention is directed to thedetermination of relative abundances of the methane isotopologues:¹²CH₄, ¹³CH₄, ¹²CH₃D and ¹³CH₃D. The apparatus and methods describedherein can be used to obtain molecular analyte ion data from a sample ofmethane. The molecular analyte ion data can then be used to determinethe isotopic compositions of the constituent components of the sampleand, thus, the relative abundances of the ¹²CH₄, ¹³CH₄, ¹²CH₃D and¹³CH₃D isotopologues in the methane sample. The relative proportions ofthe preceding methane isotopologues are a function of temperature inmethane that has achieved thermodynamic equilibrium. Thus, adetermination of the relative proportions of the preceding isotopologuesin a sample of methane can be used to measure the temperatures of originand/or storage of this component of natural gas, as an aid to theexploration and development of oil and gas deposits.

Another embodiment is directed to the determination of relativeabundances of ¹³C-bearing isotopologues of CH₃+ and C₂H₅+ ion fragmentsgenerated by ionization of propane. The foregoing data, combined withcharacterizations of the empirical constants describing fragmentationand recombination reactions in the ion source, can be used to determinethe difference in ¹³C content between the terminal and central carbonpositions of propane. This difference is predicted to be a function oftemperature in thermodynamically equilibrated propane (and thus can beused to establish the temperature of formation, as for the methaneanalysis described above). In non-equilibrated gases, this differencemay illuminate the chemical kinetic mechanisms of natural gasmaturation, and thus also aid in the exploration and development of oiland gas deposits.

Yet another embodiment of the invention is directed to the analysis ofrelative proportions of ¹³C, D and/or ¹⁸O bearing isotopologues of ionfragments generated by delivering volatile organic compounds, such asderivatized sugars, into the ion source. The foregoing data, combinedwith characterizations of the empirical constants describingfragmentation and recombination reactions in the ion source, will allowfor the characterization of isotopic fingerprints associated withdiverse sources of such compounds and thus aid in the forensic studiesof diverse organic compounds (functionally, any species that can bederivatized to create a compound that can be delivered to the ion sourcethrough a heated gas chromatographic column).

According to embodiments of the invention, a mass spectrometer includesa first ion travel path and a first introduction device configured toselect between a reference material and a first portion of an analyteand introduce the selected one of the reference material or the firstportion of the analyte to a first ion source. The first ion source has afirst entrance slit having a first width. The first ion source isconfigured to convert the reference material or the first portion of theanalyte to first molecular analyte ions and to guide the first molecularanalyte ions along the first ion travel path, each of the firstmolecular analyte ions having a momentum. A first momentum filter ispositioned downstream from the first ion source and is configured toreceive the first molecular analyte ions, the first momentum filterhaving a first radius of curvature along the first ion travel path. Themomentum filter is configured to filter out second molecular analyteions from the first molecular analyte ions according to their momenta,each of the second molecular analyte ions having an energy level. Afirst energy filter is positioned downstream from the first momentumfilter and is configured to receive the second molecular analyte ions,the first energy filter having a second radius of curvature along thefirst ion travel path. The first energy filter is configured to filterout third molecular analyte ions from the second molecular analyte ionsaccording to their energy levels. The mass spectrometer also includes adetector positioned downstream of the first energy filter and configuredto receive the third molecular analyte ions. The first width of thefirst entrance slit and the first and second radii of curvature areselected to provide a mass resolution at the detector of about 30,000 orgreater.

The detector can include a single collector, and the single collectorcan be configured to detect the third molecular analyte ions. In someembodiments, each of the third molecular analyte ions has a mass thatdiffers from the masses of the other third molecular analyte ions byless than 1 atomic mass unit. The first introduction device can beconfigured to receive the first portion of the analyte as a gas phaseanalyte. The analyte can be a gas phase analyte and the firstintroduction device can include a first inlet coupled to a samplereservoir including the gas phase analyte. The reference material can bea gas phase reference material and the first introduction device caninclude a second inlet coupled to a reference reservoir configured toaccommodate the gas phase reference material. The sample reservoir canbe configured to accommodate the gas phase analyte at first pressure,the reference reservoir can be configured to accommodate the gas phasereference material at a second pressure, and the first and secondpressures can be the same. The first introduction device can beconfigured to receive the first portion of the analyte entrained in aflow of inert gas.

In some embodiments, the first momentum filter is configured to producea magnetic field, the first energy filter is configured to produce anelectric field, and the mass spectrometer is configured to vary themasses of the third molecular analyte ions detected at the detector bymaintaining the strength of the magnetic field of the first momentumfilter at a set value and varying the strength of the electric field ofthe first energy filter. The masses of the third molecular analyte ionsdetected at the detector can be different from one another by less thanone atomic mass unit. The masses of the third molecular analyte ionsdetected at the detector can be the same when rounded to the nearestwhole number. The mass spectrometer can be configured to vary thestrength of the electric field of the first energy filter in a set rangeto vary the masses of the third molecular analyte ions detected at thedetector in a range spanning one atomic mass unit. The mass spectrometercan be configured to vary the strength of the electric field of thefirst energy filter across the set range a plurality of times to producea plurality of spectra corresponding to the range spanning one atomicmass unit. The mass spectrometer can further include a processorconfigured to produce a model of each of the spectra, and to average themodels to produce a modeled spectrum. In some embodiments, the massspectrometer further includes a processor configured to average theplurality of spectra to produce an averaged spectrum and to produce amodel of the averaged spectrum.

The first momentum filter can be configured to produce a magnetic field.The first energy filter can be configured to produce an electric field.The mass spectrometer can be configured to vary a first set of masses ofthe third molecular analyte ions detected at the detector by maintaininga first strength of the magnetic field of the momentum filter at a firstset value and varying a strength of the electric field of the firstenergy filter. The mass spectrometer can be configured to vary a secondset of masses of the third molecular analyte ions detected at thedetector by maintaining a second strength of the magnetic field of thefirst momentum filter at a second set value and varying a strength ofthe electric field of the first energy filter.

The mass spectrometer can be configured to vary the first strength ofthe electric field of the first energy filter in a first set range tovary the first set of masses of the third molecular analyte ions of thethird output detected at the detector in a first range spanning oneatomic mass unit. The mass spectrometer can be configured to vary thesecond strength of the electric field of the first energy filter in asecond set range to vary the second set of masses of the third molecularanalyte ions detected at the detector in a second range spanning oneatomic mass unit.

Embodiments of the present invention are also directed to a system foranalyzing an analyte, the system including a first mass spectrometer asdescribed above and a second mass spectrometer. The second massspectrometer includes a second ion travel path and a second ion sourcehaving a second entrance slit having a second width. The second ionsource is configured to convert a second portion of the analyte tofourth molecular analyte ions and to guide the fourth molecular analyteions along the second ion travel path, each of the fourth molecularanalyte ions having an energy level. A second energy filter ispositioned downstream from the second ion source and is configured toreceive the fourth molecular analyte ions, the second energy filterhaving a third radius of curvature along the second ion travel path. Thesecond energy filter is configured to filter out fifth molecular analyteions from the fourth molecular analyte ions according to their energylevels, each of the fifth molecular analyte ions having a momentum. Asecond momentum filter is positioned downstream from the second energyfilter and is configured to receive the fifth molecular analyte ions,the second momentum filter having a fourth radius of curvature along thesecond ion travel path. The second momentum filter is configured tofilter out sixth molecular analyte ions from the fifth molecular analyteions according to their momenta. The second mass spectrometer alsoincludes a detector array positioned downstream of the second momentumfilter and configured to receive the sixth molecular analyte ions. Thesecond width and the third and fourth radii of curvature are selected toprovide a second mass resolution at the detector array of about 20,000or greater. The system can include a processor configured to processfirst molecular analyte ion data from the first mass spectrometer andsecond molecular analyte ion data from the second mass spectrometer. Theprocessor can include a first processor configured to process the firstmolecular analyte ion data and a second processor configured to processthe second molecular analyte ion data.

Aspects of embodiments of the invention are also directed toapplications of the mass spectrometer. For example, according toembodiments of the invention, a method of identifying a potentialoil-field includes analyzing a sample from a target field using anembodiment of the system described herein to obtain molecular analyteion data, where the sample includes the analyte. The method furtherincludes analyzing the molecular analyte ion data to obtain an isotopiccomposition of at least a portion of the analyte. The isotopiccomposition of the analyte is used to determine relative proportions ofat least a portion of isotopologues in the sample. The relativeproportions of the isotopologues of the sample are compared to adatabase to determine a property of the sample, such as the temperatureof origin (e.g., temperature of formation) and/or temperature of storageof the sample. The temperature of origin (e.g., temperature offormation) and/or temperature of storage of the sample can be used inconjunction with other information to decide whether or not to drill atthe target field. In some embodiments, the analyte is a hydrocarbon,such as methane, ethane, propane, butane, pentane, hexane, or the like.

According to another embodiment of the invention, a method of analyzinga drug or drug metabolite includes analyzing a sample of the drug ordrug metabolite using an embodiment of the system described herein toconvert the drug or drug metabolite to molecular analyte ions and toobtain molecular analyte ion data, where the sample includes theanalyte. The method also includes analyzing the molecular analyte iondata to obtain an isotopic composition of at least a portion of the drugor drug metabolite. The method further includes comparing the isotopiccomposition obtained for the drug or drug metabolite to a database ofisotopic compositions. The correlation between the isotopic compositionobtained for the drug or drug metabolite and the database of isotopiccompositions can be used to determine a property of the drug or the drugmetabolite and is useful in the forensic study of diverse organiccompounds.

According to embodiments of the invention, a method of determining anamount of an anthropogenic contribution to an atmospheric concentrationof an atmospheric gas includes analyzing a sample of the atmospheric gasusing an embodiment of the system described herein to obtain molecularanalyte ion data, where the sample includes the analyte. The methodfurther includes analyzing the molecular analyte ion data to obtain anisotopic composition of at least a portion of the analyte. The methodalso includes comparing the isotopic composition obtained for theanalyte to a database of isotopic compositions. The correlation betweenthe isotopic composition obtained for the analyte and the database ofthe isotopic compositions can be used to determine the amount of theanthropogenic contribution to the atmospheric concentration of theatmospheric gas.

According to another embodiment of the invention, a method fordiagnosing or treating a disease includes analyzing a sample from apatient using an embodiment of the system described herein to obtainmolecular analyte ion data, where the sample includes the analyte. Themethod further includes analyzing the molecular analyte ion data toobtain an isotopic composition of at least a portion of the analyte. Themethod also includes comparing the isotopic composition obtained for theanalyte to a database of isotopic compositions. The correlation betweenthe isotopic composition obtained for the analyte and the database ofthe isotopic compositions can be used to determine a disease diagnosisor disease treatment protocol.

According to another embodiment of the invention, a method fordetermining a prior temperature of a sample includes analyzing thesample using an embodiment of the system described herein to obtainmolecular analyte ion data, where the sample includes the analyte. Themethod further includes analyzing the molecular analyte ion data toobtain an isotopic composition of at least a portion of the analyte. Themethod also includes comparing the isotopic composition obtained for theanalyte to a database of isotopic compositions. The correlation betweenthe isotopic composition obtained for the analyte and the database ofisotopic compositions can be used to determine the prior temperature ofthe sample.

Aspects of embodiments of the invention are also directed to methods fordetermining the isotopic composition of an analyte in a sample. Forexample, according to embodiments of the invention, a method fordetermining the isotopic composition of an analyte in a sample includesconverting a first portion of the analyte to first molecular analyteions using a first ion source of a first mass spectrometer. The methodfurther includes filtering out second molecular analyte ions from thefirst molecular analyte ions according to their momenta, and filteringout third molecular analyte ions from the second molecular analyte ionsaccording to their energy levels. The method also includes detecting twoor more of the third molecular analyte ions of the third output at amass resolution of about 30,000 or greater to produce first molecularanalyte ion data. The method further includes analyzing the firstmolecular analyte ion data to determine an isotopic composition of atleast a portion of the analyte.

In some embodiments, the two or more of the third molecular analyte ionshave respective masses that are the same when rounded to the nearestwhole number, and the first molecular analyte ion data comprises aseparate, mass resolved signal for each of the two or more of the thirdmolecular analyte ions. In some embodiments, the two or more of thethird molecular analyte ions have respective masses that differ by lessthan one atomic mass unit, and the first molecular analyte ion dataincludes a separate, mass resolved signal for each of the two or more ofthe third molecular analyte ions. In some embodiments, the third outputincludes two or more first molecular analyte ion beams, and thedetecting the two or more of the third molecular analyte ions includesscanning at least two of the first molecular analyte ion beams across asingle detector. The third molecular analyte ions of each of the two ormore first molecular analyte ion beams can have masses that differ fromone another by less than about 1 atomic mass unit. The first portion ofthe analyte can be introduced into the mass spectrometer as a continuousflow prior to converting the first portion of the analyte to the firstmolecular analyte ions. The first portion of the analyte can beintroduced into the mass spectrometer as a time-resolved pulse prior toconverting the first portion of the analyte to the first molecularanalyte ions. The analyte can include two or more analyte isotopologues,analyte isotopomers or mixtures thereof. The analyzing can furtherinclude determining the molecular position of at least one isotope in atleast one of the analyte isotopologues or the analyte isotopomers.

In some embodiments, the method further includes converting a secondportion of the analyte to fourth molecular analyte ions using a secondion source in a second mass spectrometer. The method further includesfiltering out fifth molecular analyte ions from the fourth molecularanalyte ions according to their energy levels, and filtering out sixthmolecular analyte ions from the fifth molecular analyte ions accordingto their momenta. The method also includes detecting two or more of thesixth molecular analyte ions at a second mass resolution of about 20,000or greater to produce second molecular analyte ion data. The methodfurther includes analyzing the second molecular analyte ion data todetermine an isotopic composition of at least a portion of the analyte.

According to another embodiment of the invention, a method ofidentifying a potential oil-field includes determining an isotopiccomposition of a sample from a target field according to one of themethods described herein, where the sample includes the analyte. Themethod further includes using the isotopic composition to determinerelative proportions of at least a portion of the isotopologues of theanalyte in the sample. The relative proportions of the isotopologues ofthe sample are compared to a database to determine a property of thesample, such as the temperature of origin (e.g., temperature offormation) and/or temperature of storage of the sample. The temperatureof origin (e.g., temperature of formation) and/or temperature of storageof the sample can be used in conjunction with other information todecide whether or not to drill at the target field. In some embodiments,the analyte is a hydrocarbon, such as methane, ethane, propane, butane,pentane, hexane, or the like.

According to another embodiment of the invention, a method of analyzinga drug or drug metabolite includes determining an isotopic compositionof a sample of the drug or the drug metabolite according to one of themethods described herein, where the sample includes the analyte. Themethod also includes comparing the isotopic composition obtained for thedrug or drug metabolite to a database of isotopic compositions. Thecorrelation between the isotopic composition obtained for the drug ordrug metabolite and the database of isotopic compositions can be used todetermine a property of the drug or the drug metabolite and is useful inthe forensic study of diverse organic compounds.

According to another embodiment of the invention, a method ofdetermining an amount of an anthropogenic contribution to an atmosphericconcentration of an atmospheric gas includes determining an isotopiccomposition of a sample of the atmospheric gas according to one of themethods described herein, where the sample includes the analyte. Themethod further includes comparing the isotopic composition obtained forthe analyte to a database of isotopic compositions. The correlationbetween the isotopic composition obtained for the analyte and thedatabase of the isotopic compositions can be used to determine theamount of the anthropogenic contribution to the atmosphericconcentration of the atmospheric gas.

According to another embodiment, a method for diagnosing or treating adisease includes determining an isotopic composition of a sample from apatient according to one of the methods described herein, where thesample includes the analyte. The method further includes comparing theisotopic composition obtained for the analyte to a database of isotopiccompositions. The correlation between the isotopic composition obtainedfor the analyte and the database of the isotopic compositions can beused to determine a disease diagnosis or disease treatment protocol.

According to another embodiment of the invention, a method ofdetermining a prior temperature of a sample includes determining anisotopic composition of the sample according to one of the methodsdescribed herein, where the sample includes the analyte. The methodfurther includes comparing the isotopic composition obtained for theanalyte to a database of isotopic compositions. The correlation betweenthe isotopic composition obtained for the analyte and the database ofisotopic compositions can be used to determine the prior temperature ofthe sample.

In another embodiment, a mass spectrometer

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will bebetter understood by reference to the following detailed descriptionwhen considered in conjunction with the accompanying drawings, brieflydescribed below.

FIG. 1 is a schematic top view of a first mass spectrometer according toone embodiment of the invention.

FIG. 2 is a chart showing schematic, time resolved streams of areference material and an analyte.

FIG. 3 is schematic view of a mixture of an inert gas and an analyte.

FIG. 4 is graph showing a mass spectrum obtained using a first massspectrometer according to an embodiment of the present invention.

FIG. 5 is graph showing the precision of measurements of the intensityratios of two of the ion beams illustrated in FIG. 4, made using a firstmass spectrometer according to an embodiment of the present invention.

FIG. 6 is a schematic chart showing cycles of mass spectra obtainedusing a first mass spectrometer according to an embodiment of thepresent invention.

FIG. 7 is a graph, including a close-up view, showing signal intensitiesgenerated when pulses of methane are introduced into a first massspectrometer according to an embodiment of the present invention.

FIG. 8 is a pair of graphs in which the upper graph is a time series ofmeasurements of the ratios of two peaks at 17 amu for methane, whereeach relatively flat portion of the time trace samples either the sampleor standard and the spikes are the ratios measured during the intensitydips shown in FIG. 7, and the lower graph shows averages across the flatparts of each block of data and compares each sample period to thebracketing standard periods.

FIG. 9 is a graph showing analysis of a mass spectrum obtained using afirst mass spectrometer according to an embodiment of the presentinvention.

FIG. 10 is a graph showing analysis of a mass spectrum obtained using afirst mass spectrometer according to an embodiment of the presentinvention.

FIG. 11 is a graph showing analysis of a mass spectrum obtained using afirst mass spectrometer according to an embodiment of the presentinvention.

FIG. 12 is a graph, including a close-up view, showing a mass spectrumobtained using a second mass spectrometer according to an embodiment ofthe present invention.

FIG. 13 is a graph showing a mass spectrum obtained using a second massspectrometer according to an embodiment of the present invention.

FIG. 14 is a graph showing a schematic mass spectrum corresponding touse of a second mass spectrometer according to an embodiment of thepresent invention.

FIG. 15 is a graph showing a schematic mass spectrum of the same two ionbeams illustrated in FIG. 14, but corresponding to use of a first massspectrometer according to an embodiment of the present invention.

FIG. 16 is a graph showing a two-dimensional composition space foranalyzing data acquired according to embodiments of the presentinvention.

FIG. 17 is a graph illustrating an embodiment of a method in which ameasurement of the composition of a sample using an embodiment of thefirst mass spectrometer and a measurement of the composition of the samesample using an embodiment of the second mass spectrometer can becombined to determine the concentrations of ¹³C and D containingisotopologues in a sample.

FIG. 18 is a graph illustrating the locations of three representativesamples in the two-dimensional composition space illustrated in FIG. 16.

FIG. 19 is a graph showing the results of analyses of the threeindependently known gases from FIG. 18, using an embodiment of methodsaccording to the present invention.

FIG. 20 is a graph showing another embodiment of a two-dimensionalcomposition space for analyzing data acquired according to embodimentsof the present invention.

FIG. 21 is a graph showing a mass spectrum obtained using a first massspectrometer according to an embodiment of the present invention.

FIG. 22 is a graph showing the results of analyses of the threeindependently known gases from FIG. 18, using an embodiment of methodsaccording to the present invention where both sets of contours aremeasured with the first mass spectrometer and the ¹³C/¹²C ratio isanalyzed using features of the mass spectrum of FIG. 21.

FIG. 23 is a graph, including a close-up view, showing a mass spectrumobtained using a second mass spectrometer according to an embodiment ofthe present invention.

FIG. 24 is a graph showing a mass spectrum obtained using a first massspectrometer according to an embodiment of the present invention.

FIGS. 25 and 26 are graphs illustrating how measurements according toembodiments of the present invention can be combined to constrain thetemperature of internal isotopic equilibration of propane.

FIG. 27 is a schematic top view of a mass spectrometer according toanother embodiment of the invention.

FIG. 28 is a schematic view of a sample introduction system for a massspectrometer according to one embodiment of the invention.

FIG. 29 is a cutaway schematic view of an ion source and entrance slitaperture for a mass spectrometer according to one embodiment of theinvention.

FIG. 30 is a cutaway schematic view of a detector array for a massspectrometer according to one embodiment of the invention.

FIG. 31 is a flowchart showing methods for determining the isotopiccomposition of an analyte in a sample according to embodiments of theinvention.

FIG. 32 is a partial schematic view of two detectors of a detector arrayconcurrently detecting two molecular ion beams according to anembodiment of the invention.

FIGS. 33A-D are partial schematic views showing two molecular ion beamsbeing scanned across a single detector; FIG. 33E is a graph showing theresultant mass spectrum; and FIG. 33F is a schematic view showing thecomponents of a signal intensity used for calculating mass resolution.

FIG. 34 is a flowchart showing a component of a method for determiningthe isotopic composition of an analyte in a sample according to anembodiment of the invention.

FIG. 35 is a flowchart showing a component of a method for determiningthe isotopic composition of an analyte in a sample according to anembodiment of the invention.

FIG. 36 is a flowchart showing a component of a method for determiningthe isotopic composition of an analyte in a sample according to anembodiment of the invention.

FIG. 37 is a flowchart showing a component of a method for determiningthe isotopic composition of an analyte in a sample according to anembodiment of the invention.

FIG. 38 is a flowchart showing a component of a method for determiningthe isotopic composition of an analyte in a sample according to anembodiment of the invention.

FIG. 39 is a graph illustrating the temperature dependence of isotopeexchange reactions involving homogeneous equilibria of methaneisotopologues, including multiply substituted isotopologues.

FIG. 40 is a graph illustrating the temperature dependence of anintramolecular exchange process in propane.

FIG. 41 is a flowchart showing a component of a method for determiningthe isotopic composition of an analyte in a sample according to anembodiment of the invention.

FIG. 42 is a flowchart showing a method for determining the isotopiccomposition of an analyte in a sample according to an embodiment of theinvention.

FIG. 43 is a graph illustrating predicted equilibrium constants forisotope exchange reactions involving homogeneous equilibria of methaneisotopologues, including multiply substituted isotopologues.

FIG. 44A is a graph illustrating the peak shape generated by scanningthe mass 16 AMU analyte ion beam of methane across a single detectoraccording to an embodiment of the invention; and FIGS. 44B-G are partialschematic views showing three molecular ion beams derived from methanebeing scanned across a single detector.

FIG. 45 is a graph illustrating the external precisions of replicateanalyses of the mass 17/16 ratio of methane gas according to oneembodiment of the invention.

DETAILED DESCRIPTION

Embodiments of the present invention are directed to apparatus, systemsand methods for determining the isotopic composition (or isotopicanatomy) of an analyte, such as a volatile and/or organic molecule. Forexample, embodiments of the invention are directed to apparatus andsystems for measuring intensity ratios of molecular ions, fragment ionsand/or adduct ions, and to methods for determining abundance ratios ofisotopologues and position-specific isotopic compositions of an analytewithin a sample or samples. Quantitative analysis of the relativeabundances of isotopologues of molecules can be accomplished through:(1) high-resolution, multi-collector mass spectrometric analysis ofmolecular ions, fragment ions and/or adduct ions of such molecules(e.g., the analyte) produced by gas-source electron impact ionization;(2) calibration of a variety of relevant analytical biases throughcomparison of the data obtained for the sample (e.g., the analyte) withdata obtained for appropriately prepared standards (e.g., referencematerials); and (3) reconstruction of the original molecular isotopicstructure of the analyte through integration of measured compositions ofthe various fragment species.

As used herein, the term “isotopologues” is used in its art recognizedsense and refers to molecules that have the same chemical structure, butdiffer in their isotopic composition (i.e., the isotopologues havediffering isotopic substituents). For example, CH₃D and CH₄ areisotopologues of one another. As used herein, the term“multiply-substituted isotopologue” is used in its art recognized senseand refers to a molecule that includes two or more rare isotopes. Forexample, ¹³CH₃D is a multiply-substituted isotopologue. As used herein,the term “isotopomers” is used in its art recognized sense and refers tomolecules having the same chemical composition and the same kind andamounts of isotopic substituents, but differ in the molecular positionsof at least some of the atoms (e.g., the positions of the isotopicsubstituents). For example, CH₂D-CH₂—CH₃ and CH₃—CHD-CH₃ are isotopomersof one another. Isotopomers are strictly identical at any massresolution, and cannot be separated by their respective mass to chargeratios, since they have the same mass. As used herein, the term“cardinal mass” refers to the mass of an ion or molecule after roundingto the nearest whole number. Thus, two or more ions (including molecularions) having the same cardinal mass (or a single cardinal mass) wouldhave masses that each round to the same nearest whole number, eventhough the two or more ions may have absolute masses that are differentfrom one another. Ions derived from a single sample and having the samecardinal mass may be analyzed separately using a mass spectrometer onlywhen the mass resolving power of the mass spectrometer is sufficient todistinguish the small differences in mass that arise due to one ioncontaining a heavy isotope (e.g., ¹³C) and the other ion containinganother, different heavy isotope (e.g., D). As used herein, the term“data” is used in its art recognized sense and refers to quantitiesobtained using the apparatus or methods described herein and caninclude, for example, a single ion intensity, a set of ion intensities,ratios of ion intensities, a mass spectrum and/or mass spectra. As usedherein, the terms “molecular analyte ion” and “molecular analyte ions”refer to ions of chemical compounds having two or more atoms bonded toone another and, as would be understood by those of skill in the art,encompass ions of intact analyte molecules, ions of fragments of theanalyte molecules, and ions of adducts of the analyte molecules and/orits fragments.

Embodiments of the present invention are directed to the combination ofa high resolution, high precision measurement, at a single cardinalmass, of ions having high cardinal mass with a high precision, lowerresolution measurement, at two or more different cardinal masses, ofions having different cardinal masses.

Apparatus, systems and/or methods according to embodiments of theinvention can be used in earth and environmental science (e.g.,thermometry of natural compounds and developing budgets for atmosphericgases), chemistry, forensics (e.g., chemical forensics and explosivesfingerprinting), biomedical research, diagnosis and treatment ofdiseases (e.g., drug and/or drug metabolite tracking), and hydrocarbon(e.g., oil and gas) exploration. For example, embodiments of theinvention are directed to a method of identifying a potential (e.g., ahigh-potential) subsurface hydrocarbon deposit (e.g., an oil-field).

FIG. 1 is a schematic top view of a first mass spectrometer 200according to an embodiment of the present invention. The spectrometershown in FIG. 1 may be any double-focusing, single-collector sector massspectrometer capable of ionizing molecular gases and achieving massresolutions of approximately 50,000 or greater. The embodiment shown inFIG. 1 assumes a reverse Nier-Johnson geometry. In the embodiment shownin FIG. 1, the first mass spectrometer 200 includes a first ion travelpath along a first ion source 216, a first entrance slit 239, a firstmomentum filter 222 (e.g., a magnetic sector), and a first energy filter228 (e.g., an electrostatic analyzer or “ESA”) configured to providemolecular analyte ions to a detector 234. The first mass spectrometer200 can be configured to provide a first mass resolution (which isdescribed in more detail below) of 30,000 or greater (e.g., 49,000 orgreater) at the detector 234 by sequentially arranging the firstentrance slit, the first momentum filter and the first energy filter,and by appropriately selecting a first width of the first entrance slit,and a first radius of curvature of the first momentum filter, and asecond radius of curvature of the first energy filter.

The mass resolution achieved by a mass spectrometer according toembodiments of the invention is generally proportional to the separationdistance between two ion beams that the mass spectrometer can achievefor ion beams that include respective ions having masses that aredifferent from one another. In embodiments of the first massspectrometer, the separation distance between the ion beams iscontrolled by the radii of curvature of the ion beams as they passthrough the first momentum filter and first energy filter (e.g., theelectrostatic analyzer), and inversely proportional to the width of eachion beam, which is proportional to the first width of the first entranceslit. Additionally, the highest mass resolutions can be achieved througha double focusing sector mass spectrometer design, where both momentumand energy filtering occur in an analyzer including the momentum filterand the energy filter. The first momentum filter and the first energyfilter work together in that the first energy filter images the focalpoint of the first momentum filter. Thus, according to embodiments ofthe first mass spectrometer, the ions are filtered by momentum (e.g., bythe momentum filter) prior to being filtered by energy (e.g., by theenergy filter). Accordingly, the first momentum filter has dimensionsthat are consistent with the creation of a double-focusing condition atthe detector, given the momenta of the ions as they exit the first ionsource and the first radius of curvature of the first momentum filter.For example, in embodiments of the first mass spectrometer, first massresolutions in the range of about 2,000 to about 100,000 (e.g., about30,000 to about 100,000; or about 49,000 to about 100,000) can beachieved if the first entrance slit has a first width of about 200 μm toabout 1 μm, respectively, the first molecular analyte ions areaccelerated to 5 keV after exiting the first ion source, the firstradius of curvature of the momentum filter is about 35 cm, and thesecond radius of curvature of the energy filter is about 50 cm.

Embodiments of the first mass spectrometer also include a source of ananalyte and a source of a reference material. For example, theembodiment of the first mass spectrometer shown in FIG. 1 also includesa sample reservoir 202 and a reference reservoir 204. In someembodiments, the analyte is a gas phase analyte, and the firstintroduction device includes a first inlet coupled to the samplereservoir 202. The sample reservoir 202 can accommodate a the gas phaseanalyte. In some embodiments, the reference material is a gas phasereference material, and the first introduction device includes a secondinlet coupled to the reference reservoir 204. The reference reservoir204 can accommodate the reference material (e.g., the gas phasereference material). For example, the sample reservoir 202 can include agas phase sample including a gas phase analyte, and/or the samplereservoir 202 can include a liquid phase sample and a vapor includingthe gas phase analyte. In some embodiments, the reference reservoirincludes a liquid phase reference material and/or the gas phasereference material. Accordingly, in some embodiments, the sample and thereference material may each be a room temperature gas, or a high vaporpressure liquid. The sample reservoir 202 can accommodate the gas phaseanalyte at a first pressure, the reference reservoir 204 can accommodatethe gas phase reference material at a second pressure, and the first andsecond pressures can be the same (or substantially the same).

The analyte described herein can be any suitable gas or volatilecompound (e.g., a volatile organic compound) that can be translatedthrough a tube (e.g., a confined tube) as a pure gas or as an analytemixed with a carrier gas (e.g., an inert gas). For example, a firstportion of the analyte can be entrained in the carrier gas. The analytecan be any analyte that can be suitably analyzed using the subjectmatter disclosed herein. For example, the analyte can be, or can bederived from, any suitable gas, volatile compound, semi-volatile liquidor sublimable solid. For example, volatile compounds can include anyorganic compound that can be suitably measured or analyzed in the massspectrometer, such as, but not limited to alkanes (e.g., n-alkanes),oxygenates, aromatic compounds, heteroaromatic compounds, cycliccompounds, heterocyclic compounds, and the like

Additionally, the analyte can be derived from a sample that isunsuitable for analysis in the mass spectrometer, such as a non-volatileliquid organic compound or liquid or non-sublimable solid. For example,a sample that is otherwise incapable of being analyzed in the massspectrometer can be converted into an analyzable sample by preparing ananalyte that is a derivative or reaction product of the sample and thatis capable of being analyzed in the mass spectrometer, and thereby thederivative or reaction product can be used as a proxy for the samplethat would be otherwise unsuitable for analysis or measurement. Theanalyte can be any suitable compound that can be introduced into thefirst mass spectrometer 200.

The analyte and the reference material can be provided to a firstintroduction device 212 (e.g., a changeover block), which can selectbetween the reference material and a first portion of the analyte andintroduce the selected one of the reference material or the firstportion of the analyte to the first ion source. For example, the analyteand/or the reference material can be provided to the first introductiondevice 212 in the gas phase via conduits 206 and 208 (e.g.,capillaries), respectively, for example, as a viscous bleed. When thegas phase analyte and the gas phase reference material are provided tothe first introduction device 212 from the above-described reservoirs(e.g., as a viscous bleed), the quantity of the analyte or the referencematerial provided to the first introduction device has little or novariation over time, and stable and time invariant results can beobtained.

In some embodiments, the analyte and/or the reference material aresupplied to the first introduction device entrained (or mixed) in a flowof an inert carrier gas (e.g., a helium carrier gas), for example in aneffluent from a gas chromatograph or a liquid chromatograph. Forexample, in FIG. 1 the analyte and/or the reference material can beprovided to the first introduction device 212 via a conduit 210, whichcan also be coupled to a gas chromatograph and/or a liquidchromatograph. In some embodiments, the first mass spectrometer caninclude a plurality of conduits 210 for transmitting a plurality ofcarrier gas streams, and the first mass spectrometer can further includea valve for selecting between the plurality of carrier gas streams.

Carrier gas streams (or flows) are useful for analytes and/or referencematerials having vapor pressures too low to be introduced into the firstintroduction device as a gas, and/or for analytes and/or referencematerials that have been separated by a gas chromatograph or liquidchromatograph prior to being analyzed with the apparatus, systems ormethods disclosed herein. When the analyte and/or reference material areprovided to the first introduction device entrained in a carrier gasflow, the mixing ratio of the analyte to the carrier gas and the mixingratio of the reference material to the carrier gas can be held constant(or generally constant). Variations in the mixing ratios result inincreased errors in the data obtained from the carrier gas flows,relative to data obtained from the gas phase analyte and the gas phasereference material. The analyte or the reference material can beprovided to the first introduction device 212 as a time resolved pulse.For example, FIG. 2 is a graph showing schematic, time resolved pulsesof reference material entrained in carrier gas 4 bracketing a timeresolved pulse of analyte entrained in a carrier gas flow 2. Asdescribed in more detail below, measurements of the analyte can bebracketed by measurements of the reference material to correct forvarious errors. In FIG. 2, the peak shapes shown are schematic. Inactual analyses, the flow rate of gas to the ion source will becontrolled such that several seconds of relatively stable signal isobserved at the middle of each peak. For example, FIG. 7 includes datafrom actual analyses.

High molecular weight and/or low vapor pressure materials (e.g., lowboiling point liquids) can be analyzed at low temperature (e.g., roomtemperature) using an inert gas to push the analyte into the firstintroduction device. For example, such an analyte can be cryofocused (orcryopumped) into an evacuated container (e.g., a tube under vacuum) byconnecting the container, which is under vacuum, to another containerincluding a sample. The container can then be cooled (e.g., bycontacting it with liquid nitrogen or another cold material) to condense(or freeze) the analyte in the container. The container can then bedisconnected from other container, filled with an inert gas, and warmedto a higher temperature (e.g., room temperature). The analyte and theinert gas can then be mixed (or thoroughly mixed) and the mixture can beallowed to expand into yet another container (e.g., a bellows). Theinert gas can then “push” the analyte through a conduit to the firstintroduction device. For example, FIG. 3 shows an analyte 6 mixed withan inert gas 7 in a container 8 (e.g., a bellows) being transmittedthrough a conduit 9 (e.g., a capillary) to the first introduction device212.

In the embodiment shown in FIG. 1, the first introduction device 212 isconfigured to provide the first portion of the analyte and the referencematerial to the first ion source 216 via a conduit 214. In someembodiments, the sample reservoir 202, reference reservoir 204, conduit206, conduit 208, conduit 210, first introduction device 212, and/orconduit 214 can be heated to facilitate introduction of the analyteand/or reference material to the first ion source 216.

The first ion source can be any suitable ion source used for massspectrometry, and can be the second ion source described in more detailbelow with respect to a second mass spectrometer. In some embodiments,the first mass spectrometer includes an inlet system and firstintroduction device of a gas source mass spectrometer (e.g., an inletsystem and changeover block from a THERMO DELTA V mass spectrometer,available from Thermo Fisher Scientific, Inc., Waltham, Mass.) modifiedto be coupled to an ion source of a reverse geometry, single collectorgas source mass spectrometer capable of achieving extremely high massresolutions such as 100,000 (e.g., a THERMO DFS mass spectrometer,available from Thermo Fisher Scientific, Inc., Waltham, Mass.). Forexample, in one embodiment of the first mass spectrometer, the inletsystem and changeover block from a THERMO DELTA V mass spectrometer wascombined with a THERMO DFS mass spectrometer by connecting an outlet ofthe changeover valve block of the THERMO DELTA V mass spectrometer to anaperture (e.g., a small aperture) on a side of the ion source of theTHERMO DFS mass spectrometer. In that embodiment, the connection wasmade through a vacuum housing of the THERMO DFS mass spectrometer usinga connector that abuts the side of the ion source of the THERMO DFS massspectrometer (e.g., the connector ends at a point flush with the sidehaving the aperture). The connector may be a stainless steel tube (e.g.,a ⅛″ stainless steel tube) machined to connect the changeover valveblock of the THERMO DELTA V mass spectrometer and the aperture on theside of the ion source of the THERMO DFS mass spectrometer.

In FIG. 1, the first ion source 216 is configured to convert the firstportion of the analyte (or other materials, such as the variousreference materials described below) to ions (e.g., first molecularanalyte ions). The first ion source 216 produces the ions as a firstoutput (e.g., the first molecular analyte ions). As the ions exit thefirst ion source 216, they encounter the first entrance slit 239, whichcan be included as a component of the first ion source 216 or can beseparately connected to the first ion source. The first entrance slit239 can be configured to guide the first molecular analyte ions alongthe first ion travel path.

The first momentum filter 222 can be positioned along the first iontravel path downstream from the first entrance slit 239 and can beconfigured to receive the first molecular analyte ions via a conduit218. The first momentum filter can have a first radius of curvaturealong the first ion travel path and can be configured to filter outsecond molecular analyte ions from the first molecular analyte ionsaccording to their momenta and produce a second output of molecularanalyte ions. The first momentum filter can be any suitable device thatcan filter ions according to their momenta, such as a magnetic sector.For example, the first momentum filter can be the momentum filter of aTHERMO DFS mass spectrometer, available from Thermo Fisher Scientific,Inc., Waltham, Mass.

The first mass spectrometer can also include tuning optics to guide theanalyte ions through the first mass spectrometer. For example, a firstion focusing element 220 can be included along the first ion travel pathbetween the first ion source 216 and the first momentum filter 222(e.g., the first ion focusing element 220 can be included in the conduit218). The first ion focusing element 220 may focus the second molecularanalyte ions along the first ion travel path to the first momentumfilter 222. The first focusing element can be any suitable devicecapable of focusing the second molecular analyte ions, such as anelectrostatic or magnetic lens (e.g., a quadrupole or higher formatlens), for example, an ion focusing element of a THERMO DFS massspectrometer, available from Thermo Fisher Scientific, Inc., Waltham,Mass.

The first energy filter 228 can be positioned along the first ion travelpath downstream from the first ion source 216, the first entrance slit239, the first ion focusing element 220 and the first momentum filter222, and can be configured to receive the second molecular analyte ions,which have energy levels, via a conduit 224. The first energy filter 228can have a second radius of curvature along the first ion travel pathand can be configured to filter out third molecular analyte ions fromthe second molecular analyte ions according to their energy levels andproduce a third output of molecular analyte ions. The first energyfilter can be any suitable device that can filter ions according totheir energy levels, such as an electrostatic analyzer. For example, thefirst energy filter can be the energy filter of a THERMO DFS massspectrometer, available from Thermo Fisher Scientific, Inc., Waltham,Mass.

A second ion focusing element 226 can be included along the first iontravel path between the first momentum filter 222 and the first energyfilter 228 (e.g., the second ion focusing element 224 can be included inthe conduit 224). The second ion focusing element 226 may focus thesecond molecular analyte ions along the first ion travel path to thefirst energy filter 228. The second ion focusing element can be anysuitable device capable of focusing the second molecular analyte ions,such as an electrostatic or magnetic lens (e.g., a quadrupole or higherformat lens).

A third ion focusing element 232 can be included along the first iontravel path between the first energy filter 228 and the detector 234.The first energy filter 228 can be coupled to the detector by a conduit230, and the third ion focusing element 232 can be included in theconduit 230. The third ion focusing element 232 may focus the thirdmolecular analyte ions along the first ion travel path to the detector234. The third ion focusing element can be any suitable device capableof focusing the third molecular analyte ions, such as an electrostaticor magnetic lens (e.g., a quadrupole or higher format lens), forexample, an ion focusing element of a THERMO DFS mass spectrometer,available from Thermo Fisher Scientific, Inc., Waltham, Mass.

The detector 234 can be positioned downstream of the first energy filter228 (and the third ion focusing element 232) and can be configured toreceive the third molecular analyte ions. The detector 234 can be anysuitable device used for detecting ions, such as the detector of aTHERMO DFS mass spectrometer, available from Thermo Fisher Scientific,Inc., Waltham, Mass. The detector 234 can be a single or sole detector.

The first mass spectrometer 200 can be configured to provide the thirdmolecular analyte ions to the detector 234 at a first mass resolution(which is described in more detail below) of 30,000 or greater (e.g.,from 30,000 to 100,000; from 49,000 to 100,000; or from 80,000 to100,000). For example, the first width of the first entrance slit 239and the first and second radii of curvature of the first momentum filterand first energy filter can be selected to provide a first massresolution at the detector 234 of 30,000 or greater (e.g., 49,000 orgreater). The detector can be connected to a processor (or a firstprocessor and a second processor) 236 (e.g., a computer or computers),which can be configured to acquire data from the detector and processthe data. The processor can be a processor of a THERMO DFS massspectrometer, available from Thermo Fisher Scientific, Inc., Waltham,Mass. As described in more detail below, the processor can also beconfigured to control various features of the mass spectrometer, such asthe detector. For example, the processor 236 can be connected to any orall of the components of the first mass spectrometer 200, and can beused to control the operation of the components. In some embodiments,control of the first introduction device 212 and other peripherals bythe processor 236 facilitates standardization of measurements on thefirst mass spectrometer 200. In some embodiments, some components of thefirst mass spectrometer 200 are controlled by processors separate fromand in addition to the processor 236.

According to embodiments of the present invention, the first massspectrometer can obtain a signal at one cardinal mass by setting amagnetic field strength of the first momentum filter to a set valuecorresponding to the cardinal mass of interest, and varying an electricfield strength of the first energy filter to scan across the cardinalmass. For example, by holding the magnetic field strength of the firstmomentum filter constant (or generally constant) and varying theelectric field strength of the first energy filter, the masses of ionsthat are detected at the detector can be varied across a range of onecardinal mass (or less).

In some embodiments, the third molecular analyte ions include two ormore first molecular analyte ion beams, and the detecting the two ormore of the third molecular analyte ions includes scanning at least twoof the first molecular analyte ion beams across a single detector. FIG.4 is a graph showing a signal acquired at a cardinal mass of 17 amuusing an embodiment of the first spectrometer. The peak shapes shown inFIG. 4 are generated by repeatedly scanning the three labeled ion beamscorresponding to the respective peaks across an exit slit of the firstmass spectrometer, through subtle, cyclical adjustments of the potential(e.g., electric potential) of the first energy filter, during a periodwhen the delivery of analyte to the ion source is relatively stable overtime. The exit slit leads to the detector, and, in some embodiments,ions that pass through the exit slit are registered on a singledetector. For example, in FIG. 4, signals corresponding to ¹³CH₄,¹²CH₃D, and ¹²CH₅ were acquired by holding the magnetic field strengthof the first momentum filter generally constant and varying the electricfield strength of the first energy filter to vary the mass of the ionsdetected at the detector at a cardinal mass of 17 amu. Additionally, thesignals shown in FIG. 4 were acquired by cycling the electric fieldstrength of the first energy filter to produce a plurality of scans atthe cardinal mass of 17 amu. By acquiring a plurality of scans, datahaving high reproducibility and low error can be obtained. For example,FIG. 5 is graph showing that the ratio of ¹³CH₄ to ¹²CH₃D (as shown inFIG. 4) can be acquired with 1 standard deviation (1sd)=1%, and 1standard error (1se) could approach 1‰ after a few minutes of dataacquisition.

If the electric field strength of the first energy filter is not cycledto produce a plurality of scans, signals having relatively smallerintensities and/or signals that are close to other signals from isobaricions may be difficult to detect. Accordingly, in embodiments of thepresent invention, the first mass spectrometer is configured to vary thestrength of the first electric field of the energy filter in a set rangeto vary the masses of the third molecular analyte ions detected at thedetector in a range spanning one atomic mass unit. For example, FIG. 6is a schematic chart showing a plurality of scans (or sweeps) acquiredby cycling the electric field strength of the first energy filter overtime. In FIG. 6, each of the scans is separated by a broken line. Thescans acquired through cycling (e.g., rhythmic cycling) of the electricfield strength of the first energy filter can be averaged together(e.g., “signal-averaged”) to obtain high quality data that can bemodeled (e.g., using a “peak-fitting” algorithm, such as those describedin U.S. Provisional Application No. 61/869,461, the entire contents ofwhich are incorporated herein by reference) and then analyzed using themethods described in more detail below and/or the algorithms describedin U.S. Provisional Application No. 61/869,461. In some embodiments, thescans acquired through cycling the electric field strength of the firstenergy filter can each be modeled (e.g., using a “peak-fitting”algorithm, such as those described in U.S. Provisional Application No.61/869,461) and the resultant models can be averaged together (e.g.,“model-averaged”) to obtain high quality data that can then be analyzedusing the methods described in more detail below and/or the algorithmsdescribed in U.S. Provisional Application No. 61/869,461. Over theperiod of time illustrated in FIG. 6, the magnetic field of the firstmomentum filter is fixed (or set) and the delivery of analyte to thefirst ion source is approximately constant, but the potential (e.g.,electric potential) of the first energy filter varies cyclically over anarrow range, causing two closely adjacent ion beams (one large, theother small) to be scanned across the exit slit and measured with thedetector. Dashed lines mark the end of one cycle of first energy filterpotential adjustment and the start of another. Stacking (or adding)these periodic signals together yields a peak shape such as that shownin FIG. 4.

While the electric field of the first energy filter is being cycled, thefirst mass spectrometer can switch between the analyte (e.g., thesample), the reference material (e.g., the standard), or anotheranalyte. For example, embodiments of the first introduction device(e.g., a first introduction device having a dual inlet) can provideresults having good stability and low error, even when switching betweenthe various materials. For example, FIG. 7 is a graph, including aclose-up view of the circled portion, showing performance of anembodiment of the first introduction device of the first massspectrometer while monitoring the ¹³CH₄ signal for 10 mbar of methane inthe sample bellows (10⁻⁷ mbar at the ion source), where the e-foldingtimes were 0.6 second on fall and 8 second on rise, and the signalstability was about 2×10⁻³ (1σ, 1 sec bins). In FIG. 7, the signalintensities are generated from pulses of methane introduced into a firstmass spectrometer according to an embodiment of the present invention,where valves controlling the flow of methane from either the sample orreference standard reservoirs are cycled, alternating flow from one orthe other into the ion source. Each peak top is produced by therelatively intense, stable signal of a steady flow of one or the othergases into the first ion source, and each dip is produced when flow isinterrupted by cycling of the valves that regulate gas flow. In FIG. 7,intensity is integrated across all masses in the mass scan from FIG. 4,yielding the total ion current per scan. FIG. 8 is a pair of graphsdemonstrating the performance of an embodiment of the first introductiondevice of the first mass spectrometer for a sample/standard comparisonwhile monitoring the ¹³CH₄/¹²CH₃D ratio. The upper graph in FIG. 8 showsa time series of measurements of the ratios of two peaks at 17 amu formethane, where each relatively flat portion of the time trace sampleseither the sample or standard and the spikes are the ratios measuredduring the intensity dips in FIG. 7. The lower graph in FIG. 8 showsaverages across the flat parts of each block of data and compares eachsample period to the bracketing standard periods. The data presented inFIGS. 7 and 8 was acquired using an embodiment of the first massspectrometer that can obtain precision of ±0.4 to 2‰ and 1 s.e. (whichis common for an acquisition of about 10 minutes) across range ofanalytes, mass resolutions and peak shapes (generally ˜1-2× countingstatistics). While there may still be some variation and/or error indata obtained when switching materials (e.g., analyte and standard),such data can be discarded, if desired.

Embodiments of the first mass spectrometer can also acquire data atdifferent cardinal masses. For example, the first mass spectrometer canacquire data at one cardinal mass using generally the same methods asdescribed above, and then the first momentum filter can be set toanother magnetic field strength to acquire data at another cardinal massby varying the electric field strength of the first energy filter asdescribed above. Thus, once data has been acquired at one cardinal mass,the first mass spectrometer can transition (or hop) to detecting ions atanother cardinal mass using generally the same procedures as thosedescribed above. Because embodiments of the first mass spectrometerdetect ions (e.g., the third molecular analyte ions) at one cardinalmass at a time, comparisons between data acquired at different cardinalmasses can be made from separate, non-concurrent (or non-simultaneous)measurements. While such comparisons can be made, those comparisons havea relatively high degree of error, unless they are supplemented withdata acquired using another apparatus, such as a second massspectrometer, embodiments of which are described in more detail below.

Data collected using an embodiment of the first spectrometer can beanalyzed in a variety of ways, some of which are illustrated by FIGS.9-11. One embodiment of a method for recording the intensity of one ionbeam (e.g., one ion beam of the third molecular analyte ions) in onescan is to record the maximum intensity observed as part of the peak ofthat ion beam. For example, in FIG. 9, the data is analyzed by measuringthe maximum signal intensity detected. Another embodiment of a methodfor recording the intensity of one ion beam in one scan is to averagethe intensity observed across a portion (e.g., a range) of the peak ofthat ion beam. The portion of the peak that is averaged may generally bechosen to be the most intense and/or flattest portion of the peak. Forexample, in FIG. 10, the data is analyzed by integrating over a massrange w. Another embodiment of a method for recording the intensity ofone ion beam in one scan is to create a forward model of the shape ofthe peak, where the first entrance slit width and exit slit width andion beam intensities are model parameters, and to adjust thoseparameters to achieve the best statistical fit to the measured peakshape. For example, in FIG. 11, the broken line represents a peak shapemodel that is fit to the data. As described above, the peak shape modelcan be fit to a series of scans that have been averaged (“signalaveraging”), or the peak shape model can be fit to each of a series ofscans and the models can be averaged (“model averaging”). Measuring themaximum intensity, as shown in FIG. 9, is the least labor intensiveanalysis, but it provides relatively lower precision than the othermethods shown. Integrating over a mass range, as shown in FIG. 10,provides the best compromise of convenience and precision among themethods shown. The model averaging approach of FIG. 11 provides the mostprecise results for complex, rounded peaks. The methods of FIGS. 10 and11 generally yield similar ion intensity ratios across a range of peakshapes.

Embodiments of the present invention are also directed to systems thatinclude the first mass spectrometer disclosed herein and a second massspectrometer. U.S. patent application Ser. No. 13/656,447, filed on Oct.19, 2012, entitled HIGH-RESOLUTION MASS SPECTROMETER AND METHODS FORDETERMINING THE ISOTOPIC ANATOMY OF ORGANIC AND VOLATILE MOLECULES, theentire contents of which are incorporated herein by reference, disclosesa mass spectrometer (referred to herein as “the second massspectrometer”) capable of analyzing singly and multiply substitutedisotopologues of methane, higher order alkanes and other organicmolecules at mass resolutions (M/ΔM) as high as 27,000, which issufficient to discriminate among the complex sets of isobaricinterferences that occur for molecular ions of organic compounds (e.g.,to discriminate between ¹³CH₄, ¹²CH₃D, and ¹²CH₅, each of which have amass of 17 atomic mass units). One embodiment of the second massspectrometer is a gas source, double focusing, high resolutionmulti-collector instrument.

In some embodiments, the second mass spectrometer includes the inletsystem of a gas source isotope ratio mass spectrometer (e.g., the THERMOIRMS 253, available from Thermo Fisher Scientific, Inc., Waltham, Mass.)and an analyzer and detector array resembling those of the mostsophisticated plasma and thermal mass spectrometers (e.g., the THERMONEPTUNE, available from Thermo Fisher Scientific, Inc., Waltham, Mass.).In some embodiments, the second mass spectrometer is a dual-inlet,multi-collector instrument capable of precision and accuracy sufficientfor applied stable isotope geochemistry (e.g., ±0.01 to 0.10 ‰).

The combination of components and capabilities of embodiments of thesecond mass spectrometer, which are described in more detail below,provide a mass spectrometer capable of directly and precisely analyzingthe stable isotopic compositions of organic molecules in their originalstructural forms, rather than after chemical or thermal decomposition toH₂, CO or CO₂. For example, embodiments of the second spectrometer canconcurrently (or simultaneously) detect ions at more than one cardinalmass. Additionally, because the second mass spectrometer is capable ofanalyzing the “original structure” of an analyte (e.g., molecular ionsthat retain molecular or moiety stoichiometry of the analyte), theanalyte can be meaningfully analyzed for multiply substituted (or“clumped”) isotopic species. Furthermore, the second mass spectrometeris capable of analyzing fragments of the analyte. Because organiccompounds (e.g., the analyte) fragment in distinctive ways underelectron bombardment, the second mass spectrometer is capable ofperforming measurements on pieces or fragments of analyte molecules,which allows for reconstruction of position-specific isotopic anomaliesof the analyte. Through study of multiply substituted fragment ions thesecond mass spectrometer can be used to produce position-specificclumped isotope measurements. The capabilities of the second massspectrometer allow for new isotopic approaches to the study of organicgeochemistry, including the temperatures of generation and/or storage ofnatural gases and oils, the mechanisms of biosynthetic and maturationreactions, among other applications.

The second mass spectrometer has a demonstrated ability to measureabundances of singly and doubly substituted isotopologues of methane,ethane and propane. Such measurements enable several new geochemicaltools, including a “clumped isotope” thermometer for methane based onthe reaction: ¹²CH₃D+¹³CH₄↔¹³CH₃D+¹²CH₄, which is described in moredetail below. Clumped isotope geochemistry of organic compounds iscapable of improving the understanding of the origins of natural gases,methane biogeochemistry, and the like. The second mass spectrometer canalso precisely analyze the isotopic compositions of fragments ofpropane, permitting determination of differences in δ¹³C between centraland terminal carbon positions.

The second mass spectrometer can also measure doubly ¹³C-substitutedethane (¹³C₂H₆). Such measurement of propane and ethane serve as a modelfor mass spectrometric study of the carbon isotope anatomies ofmolecular structures of other organic compounds, which can be used tostudy temperatures of formation and storage, compositions of organicmatter in source rocks, and the mechanisms of maturation reactions, allof which are relevant to oil and gas exploration. The above-describedfeatures of the second mass spectrometer are, on their own, sufficientfor analysis of low-molecular weight alkanes. For example, simple modelsof the expected mass spectra for other target analytes can be used todevelop similar measurements of isotopologues of butane, pentane andhexane.

The second mass spectrometer can detect ions with m/z up to ˜300 atomicmass units (“amu”) and, therefore, it can be used in the study of theorganic chemistry of high-molecular weight components of oils, biomassand other organic materials.

One embodiment of the second mass spectrometer (the THERMO FISHER IRMS253-ULTRA) has sufficiently high mass resolution to fully separate ionbeams that are nearly adjacent, provided their mass is sufficiently lowand/or mass separation sufficiently large. For example, FIG. 12 is agraph showing data acquired for N20 using an embodiment of the secondmass spectrometer at medium resolution (16 μm wide entrance slit;M/ΔM=17,800). The data shown in FIG. 12 was acquired using a normalgeometry, multi-collector sector mass spectrometer capable of resolvingsome nearly isobaric isotopologues from one another, provided that theirrespective masses are relatively low and/or mass separation of theisotopologues is relatively large. In FIG. 12, the signal correspondingto ¹⁴N₂ ¹⁷O is easily discernible from the signal corresponding to¹⁴N¹⁵N¹⁶O and ¹⁵N¹⁴N, and as can be seen in the close-up view of theportion of the graph encircled with a broken line, the portion of thesignal corresponding to ¹⁴N₂ ¹⁷O that is indicated with the bracket isstatistically flat (30 BDAC units; ˜0.001 AMU) and easily discernible.

One embodiment of the second mass spectrometer is capable of measuringions having masses up to 300 amu. Even at such high masses, the massresolution of the second mass spectrometer is sufficient to discriminatebetween analytes and isobaric interferences from other molecular species(e.g., contaminants). For example, ¹³C¹²C₉H₂₂ (¹³C substituted decane;having a mass of 143.1755 amu) can be resolved from ¹²C₅O₅H₃ (aprophetic fragment ion from carbohydrate or recombination product havinga mass of 142.998 amu) using a mass resolving power of only about 800.Although contaminants that differ in chemical stoichiometry from theanalyte (or analyte ions) are relatively easier to separate from theanalyte ions, isotopologues of the same chemical species (e.g., ¹³Csubstituted decane, i.e., ¹³C¹²C₉H₂₂, and D substituted decane, i.e.,¹²C₁₀H₂₁D) are substantially more difficult to separate from oneanother. For example, FIG. 13 is a graph showing data acquired forhexane using an embodiment of the second mass spectrometer at highresolution (5 μm wide entrance slit; M/ΔM=22,500). As can be seen inFIG. 13, the signals corresponding to ¹³C¹²C₃H₉ and ¹²C₄H₈D are not wellresolved from one another, and the contributions of each of ¹³C¹²C₃H₉and ¹²C₄H₈D to the measured signal are not easily discernible. The datashown in FIG. 13 was acquired using a normal geometry, multi-collectorsector mass spectrometer that is incapable of fully or partiallyresolving some nearly isobaric isotopologues from one another when massis relatively high and/or mass separation relatively small. In caseswhere the signals corresponding to different isotopologues are not fullyresolved from one another, according to embodiments of the methodsdescribed herein, the sum of the intensities of the closely adjacentpeaks can be measured, and those intensities can be separated into theircomponent parts using data acquired from the same sample on anembodiment of the first mass spectrometer. A mass resolving power of49,000 (M/ΔM) can be used to distinguish ¹³C-substituted decane fromD-substituted or H-adducted versions of ¹²C-decane. Embodiments of thesecond mass spectrometer, however, have a mass resolving power of lessthan 49,000 (M/ΔM), and the present inventor believes that anymulti-collector, normal geometry sector mass spectrometer likely willnot be able to achieve a mass resolving power of 49,000 (M/ΔM).

In contrast to embodiments of the second mass spectrometer, embodimentsof the first mass spectrometer disclosed herein are capable of achievingmass resolutions up to 80-100,000 (M/ΔM) and thus can resolve closelyadjacent ion beams that could not be resolved with embodiments of thesecond mass spectrometer. However, if the first mass spectrometer hasonly a single detector (e.g., a sole detector), precise analyses of ionintensity ratios can be made by scanning across a very narrow range inmass, and ion intensity ratios of species that share the same cardinalmass are measured together or concurrently. Accordingly, the ¹³C and Drich forms of a high molecular weight compound such as decane can beseparated from one another using an embodiment of the first massspectrometer, but their abundance relative to ¹²C or H forms would bemeasured using some other constraint.

Embodiments of the present invention are directed to the combination ofdata from the first and second mass spectrometers, so that theircomplementary traits (multi-collection at lower resolution; singlecollection at higher resolution) can both discriminate and quantifyisotopologues of high molecular weight compounds.

Accordingly, aspects of embodiments of the present disclosure relate tothe study of high molecular mass compounds (e.g., compounds havingmolecular masses greater than approximately 50 amu) through twoseparate, but related measurements. For example, in some embodiments thesecond mass spectrometer is used to precisely measure the relativeabundances of ions (e.g., fragments ions) at each cardinal mass of ananalyte, such as a high molecular mass analyte (e.g., at each cardinalmass, all isotopologue ions having the same cardinal mass are measuredtogether or concurrently).

Accordingly, in some embodiments, in addition to analyzing an analytefrom a sample using the second mass spectrometer, an analyte from thesame sample is analyzed using an embodiment of the first massspectrometer (e.g., a single-collector, reverse geometry massspectrometer according to embodiments of the present disclosure). Forexample, the sample can be analyzed at very high mass resolving power(e.g., up to about 100,000) using the first mass spectrometer toestablish the ratios of ion beams at each cardinal mass, and therelative abundances of the combination of closely-adjacent (e.g., nearlyisobaric) ions at different cardinal masses can be precisely measuredusing the second mass spectrometer. By analyzing the sample at high massresolving power with the first mass spectrometer, the proportions of theisotopologues that contribute to the signal intensity at each cardinalmass measured with the second mass spectrometer can be identified. Usingthis information, minor species can be “peak-stripped” from the signalsmeasured with the second mass spectrometer, allowing for accuratecalculation of the abundance of one isotopic species of interestmeasured with the second mass spectrometer.

For example, FIG. 14 is a graph schematically showing a signal (or massspectrum) corresponding to use of an embodiment of the second massspectrometer to analyze ¹³C substituted decane (¹³C¹²C₉H₂₂) and Dsubstituted decane (¹²C₁₀H₂₁D), which have the same cardinal mass. Asignal such as the schematic signal shown in FIG. 14 can be obtainedwhen two closely adjacent ion beams are permitted to simultaneously passthrough an exit slit of the mass spectrometer and be collected togetherin the same detector. Because an embodiment of the second massspectrometer is a multi-collector mass spectrometer, the sum of theintensities of these two species (the two ion beams) may be comparedwith intensities of one or more ion beams at some other cardinal massmeasured concurrently (or simultaneously) on another detector. As can beseen in FIG. 14, the respective signals for ¹³C¹²C₉H₂₂ and ¹²C₁₀H₂₁D arenot mass resolved from one another and, therefore, the contributions of¹³C¹²C₉H₂₂ and ¹²C₁₀H₂₁D to the peak shown in FIG. 14 cannot beindividually attributed to ¹³C¹²C₉H₂₂ and ¹²C₁₀H₂₁D. FIG. 15, on theother hand, is a graph schematically showing a signal of the same twoion beams illustrated in FIG. 14, but corresponding to use of anembodiment of the first mass spectrometer to analyze ¹³C substituteddecane (¹³C¹²C₉H₂₂) and D substituted decane (¹²C₁₀H₂₁D). In this case,the two ion beams are resolved from one another and their relativeintensities can be measured, as in FIGS. 7 and 8. For example, as can beseen in FIG. 15, the respective signals for ¹³C¹²C₉H₂₂ and ¹²C₁₀H₂₁D aremass resolved from one another and the relative proportions of¹³C¹²C₉H₂₂ and ¹²C₁₀H₂₁D that contribute to the signal measured in thesecond mass spectrometer can be determined using the data collectedusing the first mass spectrometer. Thus, the second mass spectrometercan be used to obtain a signal that is free of contributions fromcontaminant isobars, but combines signals from isobaric isotopologues(e.g., ¹³C¹²C₉H₂₂ and ¹²C₁₀H₂₁D), and the first mass spectrometer can beused to measure the abundance ratio of the isobaric isotopologues.

For example, a ratio [A]/[B] can be solved for based on the values ofratios [A+C]/[B] and [A]/[C] in a system in which [A] is theconcentration of an isotopologue of interest, [B] is the concentrationof a reference isotopologue (e.g., an unsubstituted version of amolecule or fragment of interest), and [C] is the concentration of aninterfering species in which the signals measured for A and C using thesecond mass spectrometer are unresolved and the signals measured for Aand C using the first mass spectrometer are mass resolved. If the ratios[A+C]/[B] and [A]/[C] are accurately determined, the system is fullydefined, and the ratio [A]/[B] can be solved for explicitly. If,however, the ratios determined from first mass spectrometer and thesecond mass spectrometer include an associated error, the error willpropagate to an error in the final calculated [A]/[B] ratio that can besolved for analytically for simple cases but is more easily addressedthrough Monte Carlo simulation.

FIGS. 16-22 are charts graphically illustrating embodiments of methodsin which data from the first spectrometer and/or data from anotheranalytical tool (e.g., the second mass spectrometer) is used todetermine the isotopic composition of an analyte or sample. For example,FIG. 16 is a chart representing a two-dimensional composition spacedefined by δD_(SMOW) and δ¹³C_(PDB), which are measures of D and ¹³Cconcentration, respectively, that are common in the art. δD_(SMOW) isthe deviation of the D/H ratio from that in Standard Mean Ocean Water,whereas δ¹³C_(PDB) is the deviation of the 13C/12C ratio from that inPee Dee Belemnite. Both variables are conventionally given in units ofper mil. In FIG. 16, the Cartesian coordinates represent commonvariables for reporting concentrations of ¹³C and D isotopologues, andthe broken lines represent contours of constant measured ion intensityratios, measured according to embodiments of the present invention(e.g., one set of contours represents a measurement of the ratio of twonearly isobaric species as measured with and embodiment of the firstmass spectrometer and the second set of contours represents ameasurement of the sum of those same two species divided by theabundance of some other, normalizing species having a different cardinalmass). Taken together, the intersecting contours of FIG. 16 define asecond coordinate system that is a fully defined mathematicaltransformation of the first coordinate system. For example, in FIG. 16,the small broken lines at a generally vertical diagonal angle correspondto the constant Δ(¹³CH₄+¹²CH₃D/¹²CH₃D), and the large broken lines at agenerally horizontally diagonal angle correspond to the constantΔ(¹³CH₄/¹²CH₃D). The location of a sample in the space shown in FIG. 16depends upon the concentrations of ¹³CH₄, ¹²CH₃D, and ¹²CH₄ in thesample.

Even though ¹³CH₄ and ¹²CH₃D have the same cardinal mass of 17 amu, therespective signals corresponding ¹³CH₄ and ¹²CH₃D can be mass resolvedusing an embodiment of the first mass spectrometer, and the ratio of¹³CH₄ and ¹²CH₃D can be determined. Additionally, even though ¹²CH₄ hasa cardinal mass (16 amu) that is different from that of ¹³CH₄ and ¹²CH₃D(17 amu), the signal corresponding to ¹²CH₄ and the signal correspondingto ¹³CH₄ and ¹²CH₃D can be concurrently (or simultaneously) observedusing an embodiment of the second mass spectrometer, and the ratio of¹³CH₄ and ¹²CH₃D to ¹²CH₄ can be determined. For example, FIG. 17 is achart including the two-dimensional space shown in FIG. 16 as well asprophetic data from the first mass spectrometer and prophetic data fromthe second mass spectrometer. In FIG. 17, the large broken linescorrespond to prophetic data from the first mass spectrometer, and thesmall broken lines correspond to prophetic data from the second massspectrometer. As can be seen in FIG. 17, the two sets of data can beused (e.g., combined) to identify a point within the two-dimensionalspace illustrated in FIG. 17, from which the isotopic concentrations inthe sample being studied can be determined. In FIG. 17, the compositionsof the representative samples in the two-dimensional composition spaceare independently known based on measurements common to the art.

FIGS. 18 and 19 are charts illustrating application of theabove-described methods to methane samples A, B, and C, of which A is areference sample of known concentration and B and C are samples thatwere previously analyzed by methods common in the art to determine theirisotopic abundances. The concentrations of ¹³CH₄, ¹²CH₃D, and ¹²CH₄ inthe standards A, B, and C respectively correspond to the respectivecrosses shown in FIG. 18 (i.e., the cross for A corresponds to the knownconcentration of ¹³CH₄, ¹²CH₃D, and ¹²CH₄ in the standard A). FIG. 19 isa chart including the crosses corresponding to the isotopic compositionsof samples A, B, and C, as well as data from an embodiment of the firstmass spectrometer and data from an embodiment of the second massspectrometer. In FIG. 19, the large broken lines correspond to dataobtained from an embodiment of the first mass spectrometer, and thesmall broken lines correspond to data obtained from an embodiment of thesecond mass spectrometer. The data from the first and second massspectrometers was used to identify the dots shown in FIG. 19, whichcorrespond to the respective concentrations of ¹³CH₄, ¹²CH₃D, and ¹²CH₄in the standards A, B, and C. The isotopic concentrations of the samplesA, B, and C were determined with average errors (1se) of δ¹³C: ±0.1 ‰,and δD: ±0.9% c; and average discrepancies of δ¹³C: 0.2 ‰, and δD: 1.5‰. In FIG. 19, the intersection of contour lines representingmeasurements made with embodiments of the first and second massspectrometer lie within expected analytical uncertainties of theindependently known compositions.

Variations of the above-described methods can also be used to determinethe isotopic composition of a sample, such as the methane samplesdescribed above. If there is one relevant constraint per each unknown,linear algebra can be used to construct a method for deconvolving massspectra into component parts. For example, FIG. 20 is a chart showinganother embodiment, as in FIG. 16, in which the Cartesian coordinatesare common variables for reporting concentrations of ¹³C and Disotopologues and the broken lines represent contours of constant valuesfor independent constraints, one or both of which can be ion intensityratios measured according to embodiments of the methods of the presentinvention. In FIG. 20, the two-dimensional composition space is definedby δD_(SMOW) and δ¹³C_(PDB), in which the vertical, small broken linescorrespond to the constant δ¹³C, and the diagonal, large broken linescorrespond to the constant Δ(¹³CH₄/¹²CH₃D). By varying the methodsdescribed above, data from an embodiment of the first mass spectrometeralone, or in combination with data from an embodiment of the second massspectrometer or another device or method can be used to locate a pointwithin the two-dimensional space shown in FIG. 20 that corresponds tothe sample being measured, and from that point, the isotopic compositionof the sample (or a portion thereof) can be determined.

In some embodiments, data from the first mass spectrometer may be used,on its own, to determine ratios of isotopes of one element (e.g.,¹³C/¹²C, D/H, ¹⁵N/¹⁴N, and the like) in a sample. For example, FIG. 21shows data collected at one cardinal mass using an embodiment of thefirst mass spectrometer. In the mass spectrum shown in FIG. 21, when theprobability ratio of forming the CH₃ and CH₄ ion species is equal in thesample and standard, the ¹³C/¹²C ratio of the analyte can be determinedby comparing the ¹³CH₃ to the ¹²CH₄ ion beam and normalizing to areference standard having a known ¹³C/¹²C ratio. Using that data, theratio of ¹³C to ¹²C in the sample can be determined according to theequation ¹³C/¹²C=(¹³CH₃/¹²CH₄)×(CH₃/CH₄). For example, FIG. 22 is achart illustrating application of the above-described method to themethane samples A, B, and C, described above. FIG. 22 shows the resultsof analyses of the three independently known gases from FIG. 18, usingan embodiment of methods according to the present invention where bothsets of contours are measured with the first mass spectrometer and the¹³C/¹²C ratio is analyzed using features of the mass spectrum of FIG. 21Data from the first mass spectrometer was used to identify the dotsshown in FIG. 22, which correspond to the respective concentrations of¹³CH₄, ¹²CH₃D, and ¹²CH₄ in the standards B and C. The isotopicconcentrations of the samples B and C were determined with averageerrors (1se) of δ¹³C: ±1.1 ‰, and δD: ±0.9‰; and average discrepanciesof δ¹³C: 0.9 ‰, and δD: 0.7 ‰. In FIG. 22, the intersection of contourlines representing measurements made with an embodiment of the firstmass spectrometer lie within expected analytical uncertainties of theindependently known compositions. Such methods are most readily appliedto difference in the number of H among, fragments, adducts and hydrides.

Embodiments of the above-described methods can be used to identifyisotopes at particular positions of an isotopologue. For example,embodiments of the above-described methods can be used to create aposition-specific D/H “thermometer” for propane. FIG. 23 is a chartshowing data acquired using an embodiment of the second massspectrometer at a cardinal mass of 16 amu from a sample includingpropane. Analysis of the ratio ¹³CH₃/¹²CH₃ using the second massspectrometer can define the δ¹³C of the terminal carbon site of propane.Comparison of this result with any measurement of the δ¹³C of the fullmolecule allows for calculation of the difference in δ¹³C between theterminal and central carbon site of propane. FIG. 23 illustrates theability of an embodiment of the second mass spectrometer to resolvenearly isobaric species of fragment ions of organic molecules (e.g.,separating ¹³CH₃ from ¹²CH₄ ions generated from propane). FIG. 24 is achart showing data acquired using an embodiment of the first massspectrometer at a cardinal mass of 30 amu from the sample includingpropane. FIG. 24 illustrates the ability of an embodiment of the firstmass spectrometer to resolve ¹³C- and D-bearing isotopologues of theC₂H₅ fragment ion of propane. Accordingly, the first mass spectrometercan be used to measure the ion intensity ratios of ¹³C- and D-bearingisotopologues of fragment ions of propane. Data of the type shown inFIGS. 23 and 24 can be used to define the difference in D/H ratiobetween the terminal and central carbon sites of propane. In apopulation of propane molecules at thermodynamic equilibrium, thehydrogen atoms attached to the central carbon position should have ahigher D/H ratio than those attached to the terminal carbon positions,and this difference should be larger at low temperature and smaller athigh temperature. Thus, a measurement of the D/H ratios of fragmentsions that differ in their proportions of hydrogen at the terminal andcentral sites should define the contrast between the two sites and thusthe temperature of the population of propane molecules.

For example, FIGS. 25 and 26 illustrate how measurements according toembodiments of the present invention can be combined to constrain thetemperature of internal isotopic equilibration of propane. For example,by measuring the D concentration of two or more fragment ions thatdiffer in their proportions of hydrogen at the terminal and centralcarbon positions of the molecule, the difference in D/H ratio of thosepositions can be constrained. As noted above, this difference is largeat low temperature and small at high temperature in a population ofpropane molecules that have achieved thermodynamic equilibrium. Similarmethods can be used for multi-component, multi-method analysis of largermolecules.

According to embodiments of the present disclosure, the first massspectrometer includes a single collector that detects signals at one(e.g., a single or sole) cardinal mass by rapidly scanning across all ofthe signals produced by the component isobars, before detecting signalsat another cardinal mass. As described above, once the first massspectrometer collects a suitable spectrum at one cardinal mass, thecollector of the first mass spectrometer can switch (or jump) to anothercardinal mass and the single collector can detect signals at the other(e.g., the other single or sole) cardinal mass by rapidly scanningacross all of the signals produced by the component isobars at the othercardinal mass. While embodiments of the first mass spectrometer candetermine relative proportions of species at each cardinal mass, and cando so at a very high resolution, embodiments of the first massspectrometer do not achieve the same high precision as that of thesecond mass spectrometer (e.g., a multi-collector mass spectrometer). Insome embodiments, the relative peak heights measured using the firstmass spectrometer have errors of about 1% after standardization with areference material, but the errors may be substantially smaller. Thefirst mass spectrometer, however, is not required to have highprecision, because the measurements from the first mass spectrometer canbe used to ion-correct an already precise measurement of the total ofall species at each cardinal mass detected using the second massspectrometer.

Embodiments of the second mass spectrometer can measure ion intensityratios at two cardinal masses (e.g., measuring on the flat tops ofpeaks, where all isobars are concurrently or simultaneously observed)with an external error of 0.1 per mil or better. Indeed, attaining sucha level of precision is straightforward for an embodiment of the secondmass spectrometer that is a multi-collector gas source massspectrometer. Further, embodiments of the second mass spectrometer havemass resolving power sufficient to separate out most contaminants suchthat the signals observed using the second mass spectrometer cangenerally be assumed to include contributions isotopic isobars of thesame molecule (or its fragments). For example, in some embodiments, thesignal obtained by the second mass spectrometer may be substantiallyfree of a contribution from contaminants (or the signal may includecontributions only from isotopic isobars of the same molecule or itsfragments). On the other hand, embodiments of the first massspectrometer may be able to obtain intensity ratios for components atany one cardinal mass at a precision of about 1%. Using bracketingstandardization and repeated analyses, substantially better levels ofprecision may be obtained using the first mass spectrometer. Based onthe levels of precision described herein, the errors in measuring eachisotopic species can be calculated using the ratios [A+C]/[B], [A]/[C]and [A]/[B] described herein. For example, the errors propagated throughthe calculations can be calculated using simulations such as Monte Carlomethods. It has been found that Monte Carlo simulations of propagatederrors in the final measured isotope ratio of interest follow simplescaling relationships.

For highly abundant species (e.g., species that dominate the measuredsignal at one cardinal mass), the measured precision is generally equalto (relative error in the measurement from the first massspectrometer)/(ratio of the abundant species to less abundant species atthe cardinal mass). Such levels of precision provide good measurementsfor major species, even given conservative performance from the firstmass spectrometer. For example, if a sample of decane is analyzed usingthe second mass spectrometer and found to have a ratio of the 143 amucardinal mass to the 142 amu cardinal mass of 0.138±01.38×10⁻⁵ ‰ (with0.1 ‰ error), which is roughly equivalent to the ratio found in naturalabundance decane, and the same sample is analyzed using the first massspectrometer and found to have a ¹³C¹²C₉H₂₂/¹²C₁₀H₂₁D abundance ratio of42±0.42 (with 1% error), which is also roughly equivalent to the ratiofound in natural abundance decane, then the abundance of ¹³C¹²C₉H₂₂(i.e., the δ¹³C of the C10 molecular ion) in the sample will bedetermined with an error of 0.24 ‰ (calculated using Monte Carlotechniques based on the levels of precision described herein). Thus,precision in δ¹³C is degraded by about 0.1‰ from the error in themeasurement of the 143/142 ratio using the second mass spectrometer. Anerror of about 0.2‰ is generally within the range of experimental errorin conventional δ¹³C measurements, and so an error of about 0.24‰ isacceptable.

For minor components (e.g., species that provide a relatively minorcontribution to the measured signal at one cardinal mass), errors may belarger. In some embodiments, the errors for such species may converge onthe relative error for the least abundant species for the spectrumobtained using the first mass spectrometer. For example, Monte Carlomodeling of the decane example described above may provide an error in¹²C₁₀H₂₁D abundance of 10‰, which is equal to the 1% error that may beobtained using the first mass spectrometer on its own. In someinstances, such larger errors will hinder useful application of thesubject matter disclosed herein to species that are minor components ofa given cardinal mass and that have subtle isotopic variations.

Several methodological approaches, however, may reduce the effectsassociated with errors corresponding to minor species. For example, thefragmentation spectra of organic compounds are so complex that cardinalmasses can be selected for their relatively smaller errors associatedwith the isotopic species of greatest interest (e.g., H-poor fragmentscan be selected when δ¹³C is being determined). Additionally, resultsfor fragments that differ in relative contributions of H and C can becompared to de-convolve relative contributions of D and ¹³C at eachcardinal mass. Furthermore, the use of sample-standard comparisons onpure analytes at matched pressures, which can be achieved for the firstmass spectrometer by using a THERMO DELTA V changeover (available fromThermo Fisher Scientific, Inc., Waltham, Mass.), will reduce errors tothe ‰ range, even for minor species. Moreover, even if the measurementsare constrained to the most abundant species at each cardinal mass, thesubject matter disclosed herein will still provide a very large numberof new measurements and extend the analysis capabilities of embodimentsof the second mass spectrometer to analytes far into the high molecularmass range. While embodiments of the first mass spectrometer disclosedherein complement the capabilities of embodiments of the second massspectrometer, the first mass spectrometer is also useful for isotopicanalysis by itself, as described above. Nonetheless, in someembodiments, embodiments of the first mass spectrometer can be includedin a system that also includes an embodiment of the second massspectrometer to provide fully constrained ion correction of isotopicmeasurements of high molecular weight species.

One of the benefits of embodiments of the subject matter disclosedherein is the ability to analyze single, double and triple ¹³Csubstitutions in high-molecular-weight components of oils, includingboth proportions of these substitutions in the whole molecule andproportions of some or all of these substitutions in fragment ions. Theinformation provided by such analysis can be used to construct a “map”of ¹³C substitutions in the carbon backbones of components of oils, andcan be used to provide a high-dimensionality forensic fingerprint fordiscriminating sources of oil components, which can be used indeconvolving components of oils that have mixed sources or identifyingoils and gases that include the same components. Depending upon thephysical processes that control the carbon isotope anatomies of oilcompounds, the subject matter disclosed herein may also be useful foridentifying the biosynthetic pathways of parent compounds to oilcomponents; identifying fingerprints of thermal maturation in there-arrangement of carbon isotope anatomies; finding proxies quantifyingand correcting for biodegradation; and finding molecular sites that arerelatively susceptible to exchange or re-ordering and that take on athermodynamically controlled position-specific and/or clumped isotopecomposition and thus can be used as quantitative thermometers.

The apparatus, systems and methods disclosed herein may be used to studyrelatively volatile components of oil that are structurally similar andonly somewhat larger than the compounds described with respect to thesecond mass spectrometer in U.S. patent application Ser. No. 13/656,447,filed on Oct. 19, 2012, entitled HIGH-RESOLUTION MASS SPECTROMETER ANDMETHODS FOR DETERMINING THE ISOTOPIC ANATOMY OF ORGANIC AND VOLATILEMOLECULES. For example, the apparatus, systems and methods disclosedherein may be used to empirically characterize carbon isotope anatomiesof natural and synthetic octanes (e.g., characterize howposition-specific and clumped isotope compositions of octanes relate totheir environment and mechanism of formation).

The apparatus, systems and methods disclosed herein can also be used toanalyze isoprene (CH₂═CCH═CH₂), which has 2,304 stable isotopologues,2,295 of which are multiply substituted (i.e., “clumped”) isotopologues.As many as 33 of the isotopologues are analyzable at est. ±0.1-2 per mil(‰). Isoprene, which has a boiling point of 34° C. can be introducedinto a mass spectrometer by being pushed through a bellows using heliumas described above. Isoprene has 5 symmetrically non-equivalentisotopologues having one ¹³C; 4 symmetrically non-equivalentisotopologues having one D; 10 symmetrically non-equivalentisotopologues having two ¹³C's (4 constraints); 20 symmetricallynon-equivalent isotopologues having one ¹³C and one D (5+ constraints);and about 18-33 of those isotopologues are analyzable with usefulprecision using the apparatus, methods and systems described herein.N-hexane has 33 readily analyzable singly and doubly-substitutedtargets: 3 symmetrically non-equivalent isotopologues having one ¹³C; 3symmetrically non-equivalent isotopologues having one D; 9 symmetricallynon-equivalent isotopologues having two ¹³C's; and 18 symmetricallynon-equivalent isotopologues having one ¹³C and one D. In addition tocompounds such as octane, isoprene and hexane, the apparatus, systemsand methods disclosed herein can be used to study refractory componentsof oils (and other high molecular weight organics).

Embodiments of the apparatus, systems and methods disclosed herein canbe used in the study of temperatures of formation and/or storage ofnatural gas and oil; climate records (e.g., temperatures) from, forexample, cellulose, waxes, and/or lipids; metabolisms of extant andancient life; sources of pollutants; sources of greenhouse gases; andorigins of meteoritic organic matter; mechanisms of chemical isotopeeffects; and they can be used in criminal forensics; and biomedicalresearch and diagnosis.

As discussed above, isotopologues are compounds, such as organiccompounds, that include non-equivalent isotopic substitutions, such asthe substitution of ¹³C for ¹²C, D for H, ¹⁵N for ¹⁴N, and the like, insymmetrically non-equivalent sites or combinations of sites.Isotopologues are generally distinct from one another in their chemicaland physical properties. Thus, isotopologues generally can befractionated from one another by chemical and physical processes, suchas processes responsible for formation, consumption and/or transport ofthe isotopologues. The apparatus, systems and methods disclosed hereincan be used in the study of any process, activity, interaction and/orcondition that results in fractionation of isotopologues.

For example, the presently disclosed apparatus, systems and methods canbe used to study catalyzed reactions that result in fractionation ofisotopologues. Catalysts (e.g., enzymes) capable of catalyzing a givenreaction, but having structures that are different from one another, maydiffer in the isotopic dependence of the reactions they catalyze as aresult of structural differences between the catalysts, which may resultin differences in the distribution of isotopes in the products of thecatalyzed reactions (e.g., may result in distributions of isotopologuesthat are different from one another). For example, the apparatus,systems and methods disclosed herein can be used to study conversion(e.g., metabolic conversion) of coenzyme A and pyruvate, a metabolitederived from sugars and common to all known living organisms, to acetylcoenzyme A. The enzymes pyruvate decarboxylase and pyruvatedehydrogenase can catalyze the reaction of coenzyme A with pyruvate toform acetyl coenzyme A. Those reactions of coenzyme A with pyruvateproceed at a rate that depends upon the carbon isotope at the C2 and/orC3 position of pyruvate (shown below).

For example, the reaction of coenzyme A with pyruvate having ¹²C at theC2 and/or C3 position proceeds at a higher reaction rate than that ofthe reaction of coenzyme A with pyruvate having ¹³C at the C2 and/or C3position. The reaction rate of coenzyme A with pyruvate, however, isrelatively insensitive to the identity of the carbon isotope at the C1position of pyruvate (shown below). Thus, any process (e.g., anymetabolic process) that converts pyruvate and coenzyme A into acetylcoenzyme A and converts only a portion of the available pyruvate, willpreferentially produce acetyl coenzyme A having ¹²C at the C2 (C═O) site(shown below), but leave the C1 site of acetyl coenzyme A (shown below)relatively unmodified.

For example, a sample of acetyl coenzyme A prepared from only a portionof a sample of pyruvate will have a carbon isotopic distribution that is¹²C enriched, relative to the sample of pyruvate, at the C2 sites ofacetyl coenzyme A, but will have a carbon isotopic distribution at theC1 site of the acetyl coenzyme A that is generally equivalent to theisotopic distribution of carbon in the sample of pyruvate. The foregoingisotope effect can be thought of as an isotopic “fingerprint” of thereaction that forms acetyl coenzyme A from coenzyme A and pyruvate. Forexample, a reaction catalyzed by pyruvate decarboxylase may result infractionation (e.g., isotopic distribution) that is different fromfractionation resulting from a reaction catalyzed by pyruvatedehydrogenase. Further, the above-described differences between the C1and C2 carbon positions of fractionated isotopologues of acetyl coenzymeA also affect carbon isotope distributions in fatty acids produced fromthose fractionated isotopologues, in that the resultant fatty acids havesystematic differences in ¹³C abundances at the even and odd carbonpositions (or sites). An example fatty acid is shown below having evensites (e.g., 2) and odd sites (e.g., 1). The above-described principlescan also be used in the study of acetate and amino acids prepared fromacetyl coenzyme A.

Intramolecular isotopic differences also occur in other organiccompounds produced by catalyzed reactions, such as amino acids, sugars,vanillin and the like. While the origins of the isotopic differences inthese other compounds are less well understood than those in acetylcoenzyme A, these isotopic differences likely represent a fingerprint orisotopic signature of the biosynthetic pathways by which these moleculeswere formed and/or destroyed. Indeed, study of isotope effects onirreversible, abiological chemical reactions using highly isotopicallyenriched reagents or extreme extents of distillation (e.g., highlypurified compounds) confirms that organic reactions generally lead todistinctive patterns of isotopic substitutions in partially consumedreactants and products (e.g., products produced by reactions in whichonly a portion of the reactants are consumed, and products that havebeen partially consumed), and that these patterns can be used to infermolecular and/or atomistic mechanisms of reaction. Based on theprinciples of chemical physics underlying fractionation ofisotopologues, such as fractionation of isotopologues of acetyl coenzymeA, the apparatus, systems and methods disclosed herein can be used tostudy fractionated isotopologues of a wide variety of organic compounds.

Other apparatus, systems and methods distinguish among only a smallnumber of isotopologues and are unable to provide a record of isotopicvariations produced by organic synthesis and/or consumption reactionssufficient to make detailed inferences regarding the reaction histories(e.g., identifying specific enzymes involved) by which the isotopologueswere made or partially consumed. In contrast, embodiments of theapparatus, systems and methods disclosed herein are capable ofquantifying proportions of large numbers (e.g., tens to hundreds) ofisotopologues of a wide variety of chemical compounds, at naturalisotopic abundances and with precisions of est. 10⁻³ to 10⁻⁴, relative(e.g., standard errors on the order of 0.1 to 1 per mil in a measuredabundance ratio of two isotopologues). The capabilities of embodimentsof the apparatus, systems and methods disclosed herein have beendemonstrated for n-alkanes, acetone, acetaldehyde and isoprene. Based onthe principles of chemical physics underlying fractionation ofisotopologues and the principles described herein, the apparatus,systems and methods herein can be used in the study of many commonconstituents of living organisms (e.g., sugars, amino acids and fattyacids) and a variety of compounds introduced into the body as drugs(e.g., valproic acid, which is described in more detail below). Forexample, relatively larger, heavier, and/or less volatile chemicalcompounds can be studied using the apparatus, systems and methodsdisclosed herein by way of derivatization chemistry, to improvevolatility of the chemical compounds, and/or transfer of the chemicalcompounds through heated, purged non-reactive tubing, both of which arewell understood methods used in the study of chemical compounds.Accordingly, the apparatus, systems and methods disclosed herein presentthe first practical approach for observing large numbers ofisotopologues of diverse biomolecules (which are relatively largerand/or heavier chemical compounds). Applications of the apparatus,systems and methods disclosed herein are described in more detail below.

Environmental And Criminal Forensics

The apparatus, systems and methods disclosed herein can be used to makemeasurements of natural-abundance isotope distributions that are capableof distinguishing among sources of compounds of forensic interest, suchas, for example, environmental pollutants, illicit drugs, mislabeledagricultural products (e.g., intentionally mislabeled agriculturalproducts), and chemical and biological weapon agents. Such measurementstake advantage of the fact that compounds made by different reactionpathways, under different environmental conditions (e.g., differenttemperatures), or in different geographical locations have differentabundances of naturally occurring rare isotopes. These isotopicdifferences can be thought of as “fingerprints” that can be used todistinguish among chemical compounds from different sources.

While the above-described principles can be applied to forensic problemsusing apparatus, systems and methods other than those disclosed herein,those applications only constrain the bulk abundances of isotopes in agiven sample, irrespective of the molecular sites of substitution of theisotopes or the proportion of molecules that contain multiple isotopicsubstitutions. For this reason, such measurements provide a small numberof independent constraints on the origin of a compound, and thus provideinsufficient constraints for fully analyzing forensic problems. Forexample, a measurement of the D/H isotope ratio of hair (principallykeratin proteins of the hair) is a function of the D/H isotope ratio ofdietary water, which varies with geography and thus can be a“fingerprint” of geographic provenance of the hair.

Local waters of many different geographic locations, however, sharesimilar D/H isotope ratios, and different individuals consume water fromregional water sources differently due to differences in diet. Thus, theabove-described forensic measurements of hair are non-unique. In theexample of hair keratin, an additional constraint based on a measurementof bulk isotopic abundance, such as the ¹³C/¹²C ratio of carbon, whichdiffers between leafy plants and most grains and thus differs betweenindividuals based on their dietary preferences, could be added. Theaddition of a second, independent compositional dimension (e.g., theabove-described ¹³C/¹²C ratio of carbon) would improve the reliabilityof an isotopic forensic analysis based on such measurements. Given thediversity of dietary habits of individuals, however, even with theadditional constraint, such measurements would still not providesufficient constraint for a full forensic analysis.

Due to the very large number of isotopic variants of some chemicalcompounds, a full forensic analysis may be carried out using additionalconstraints. For example, each of the amino acids in each protein inhair keratin has a very large number of isotopic variants. Even alanine(chemical formula: C₃H₇NO₂), which is a relatively simple amino acid,has 18,432 isotopic variants. The abundance of each of these variantscan be thought of as a potentially independent dimension of acomposition space in which the locations of samples of forensic interestcan be plotted. While measurements of bulk isotopic abundance of D/Hand/or ¹³C/¹²C in a sample of alanine would be plotted in a 1 or 2dimensional composition space (e.g., a line or plane), a full analysisof all isotopologues of alanine would provide an 18,432 dimensionalvolume, each dimension of which has the potential to record isotopicdifferences resulting from differences in geographic origin, dietarysources or other factors relating to the alanine being examined. Whilecreating and using such complex isotopic data presents some challenges,experience gained from working with similarly complex genomic data canbe drawn upon to guide analysis of the isotopic data to greatly improvethe ability to distinguish among compounds having different origins.

The apparatus, systems and methods disclosed herein are demonstrablycapable of measuring dozens to hundreds of isotopologues of manychemical compounds, and in principle could be extended to many moreisotopologues. The apparatus, systems and methods disclosed hereinshould be capable of providing forensic distinctions among compounds ofdifferent origin in composition space volumes having dozens to hundredsof independent dimensions.

Oil and Gas Exploration

It is useful in petroleum exploration to identify or define the sourcesof petroleum products (e.g., the collection of organic compounds fromwhich the petroleum products are derived), the nature and extent ofprogress of reactions by which they form, and the conditions (e.g.,temperatures, pressures, etc.) of their formation and storage. Theapparatus, systems and methods disclosed herein can be used to makemeasurements that can be applied to the field of oil and gas explorationin a variety of ways. A measurement that defines the temperature offormation and/or storage of iso-octane (C₈H₁₈; an important component ofgasoline and one of the most valuable distillates of natural petroleum)is one example of such a measurement.

Hydrogen occupies a variety of symmetrically non-equivalent sites iniso-octane. Among the differences in the hydrogen atoms of iso-octaneare the number of hydrogen atoms bound to each carbon: 15 are present asgroups of 3, bound to a single carbon (the hydrogen atoms of the methylgroups); 2 are present together, bound to a single carbon (the hydrogenatoms of the “CH₂” group); and 1 is bound alone to a single carbon (thehydrogen atom of the “CH” group). A population (or sample) of iso-octanemolecules that achieves intra-molecular and inter-molecular isotopicequilibrium will distribute D unevenly among the above-describedhydrogen atom sites, concentrating D into the “CH” (or “CD”) and “CH₂”(or “CHD” or “CD₂”) groups, and H into the methyl groups. The strengthand temperature dependence of the foregoing effect is known fromexperiment and theory for some fatty acids and n-alkanes, indicatingthat the difference in D/H ratio between the “CH₂” and methyl groupsshould be a factor of ˜1.09 at subterranean, near-earth-surfacetemperatures (e.g., ˜50° C.), and decrease monotonically with increasingtemperature to a factor of ˜1.03 at the highest temperatures ofpetroleum genesis (e.g., ˜200° C.).

The apparatus, systems and methods disclosed herein are capable ofmeasuring D/H ratios of fragment ions of organic compounds with aprecision of ˜0.001, relative (e.g., a standard error of 1 per mil).Different fragment ions contain different proportions of hydrogen atomsfrom different molecular sites and, thus, by comparison of the D/Hratios of two or more fragment ions, the temperature of equilibration ofiso-octane can be defined with a precision on the order of a fewdegrees. It has not yet been established whether natural iso-octaneforms in isotopic equilibrium with respect to the foregoing property, orwhether the temperatures it records are those of its formation orstorage (or perhaps both, depending on circumstances). Nevertheless, theabove-described analysis of iso-octane is just one representativeexample of a large number of possible analyses related to the apparatus,systems and methods disclosed herein.

Personalized Medicine

The apparatus, systems and methods disclosed herein can be used inmonitoring and analysis of drug budgets. Many drugs introduced into thehuman body are destroyed and excreted by diverse mechanisms. Forexample, valproic acid (C₈H₁₆O₂), which is often used as an anti-seizuremedication (e.g., for treatment of epilepsy) and, less often, as a moodstabilizer for bipolar disorder, is destroyed by a chemical reaction ina patient's mitochondria, by reaction with one or more of six differentenzymes in the patient's liver, or it is directly excreted from thepatient's body. As such, the body of a patient taking this drug can bethought of as a living chemical rector having a quasi-steady-statebudget of valproic acid that is characterized by one source term (theingested drug) and eight “sink” terms (the various mitochondrial andenzymatic destruction pathways and excretion).

Many drugs have associated side effects that result, directly orindirectly, from the metabolic destruction of the drug rather thanthrough action of the intact drug. For example, valproic acid interfereswith liver function, reduces the ability of the liver to metabolizedietary fatty acids, possibly leading to liver damage or death,pancreatitis and/or extreme weight gain. These side effects likelyresult from the chemistry of valproic acid destruction in the liver. Theseverity of side effects on any given patient are difficult to predict,and generally can be monitored only by observing negative symptoms inthe patient.

It may be possible to characterize an individual patient's tolerance fora drug, such as valproic acid, prior to the emergence of negative sideeffects in the patient by analyzing the state of the “budget” for thatdrug in the patient (e.g., by analyzing the balance of sink processesthat the patient's body uses to destroy or excrete the drug). Becauseenzymes of a given type may vary in their structure and reactivity, afull characterization of an individual patient's budget for a drug,including isotopic “fingerprints” associated with each sink term, maydetect and characterize any pathological abnormalities in theefficiencies of one or more of the enzymes that a patient's body uses tometabolize the drug.

Previous studies using isotopically labeled drug compounds, such asvalproic acid, have identified possible sink processes for thedestruction of the drug and indicated their typical or average relativerates. More information is needed, however, to fully characterize thestate of an individual patient's chemical budget of a drug at any giventime. The measurement of a large number of isotopologues of a drug (forexample, measurement of several dozen isotopologues may be sufficient)may provide an isotopic fingerprint useful for characterizing the stateof a patient's chemical budget for that drug, provided that the isotopiccomposition of the drug is known prior to being administered (e.g., bymeasuring the isotopic composition of the drug prior to ingestion by thepatient). Each of the sink processes for the destruction of the drug mayhave associated with it a set of distinctive isotopic fractionationsthat result from the isotope dependence of rates of enzymatic reaction.While some of these isotope effects are unknown at present, they may bedetermined through experimental study of in vitro reaction of the drug(e.g., valproic acid) with the relevant isolated enzymes. If each sinkprocess is unique with respect to an isotopic fingerprint (e.g., thedependence of reaction rate on isotopic substitutions in valproic acid),then a measurement that constrains proportions of 9 or moreisotopologues in both the consumed drug (e.g., the medicinal dose takenby the patient) and the quasi-steady-state blood level of the patientwould be sufficient to characterize the proportions of all 8 major sinkprocesses and, thus, the balance of that patient's budget for that drug.Measurement of more than 9 isotopologues would over-constrain the budget(e.g., provide surplus information) and mitigate against any loss ofinformation that results from too close of a similarity in some isotopeeffects associated with multiple sink processes (e.g., “degeneracy” inthe family of constraints).

Similar principles could be used to constrain (or analyze) the budgetsof a wide range of drug compounds in individuals (e.g., people oranimals). The foregoing principles can be put into practice for any onecase by addressing the following technical matters: (1) obtainingconcentration analyzable quantities (typically micrograms to milligrams)of the compound of interest from blood or tissue samples; (2) chemicalpreparation of the analyte to increase volatility (e.g., derivatizationof valproic acid to methylate or perfluorinate the hydroxyl site ofvalproic acid), if desired; and/or (3) experimental characterization ofthe end-member reactions involved in that drug's biochemistry (e.g.,determination of the set of fractionations of proportions of allmeasured isotopologues associated with reaction with each of the “sink”enzymes).

The apparatus, systems and methods disclosed herein can be used in thediagnosis of metabolic disease. For example, the principles describedabove could be extended to biochemical compounds other than drugs,including a large number of sugars, amino acids, fatty acids and othermetabolites. Budgets of these biochemical compounds in individuals(e.g., people or animals) may be identified through characterization ofa number isotopic constraints (e.g., measurements of a number ofisotopologues) equal to or greater than the number of major source andsink processes in the budget. Similarly to the above-described drugbudgets, the possibility of redundancy or close similarity in someisotope effects among two or more source and/or sink terms may make itadvantageous to substantially (or significantly) over constrain thebudget (e.g., by measuring ˜2 dozen isotopologues of a compound that hasa budget characterized by 5-10 source and/or sink terms).

The above-described methods may be particularly useful for the diagnosisand detailed characterization of metabolic diseases, such as thoseinvolving carbohydrate metabolism (e.g., diabetes) or amino acidmetabolism (e.g., phenylketonuria). For example, type 2 diabetes ischaracterized by a failure to metabolize glucose (or a lessened abilityto metabolize glucose), despite the presence of an adequate abundance ofinsulin in the body. This disorder is believed to involve a defect (ordefects) in an insulin receptor, but the exact mechanism of the disorderis unclear. A measurement that characterizes the isotopic “budget” ofblood glucose could provide insight into the balance of sink reactions(or terms) for blood glucose (e.g., in response to catabolic and/oranabolic hormones) and the molecular or atomistic mechanisms of thosereactions (e.g., by characterizing the pattern of isotope effectsassociated with the consumption or destruction of blood glucose). Suchmeasurements may reveal the existence of unrecognized sub-types of type2 diabetes, and provide for monitoring and characterizing the disease inindividuals.

Environmental Chemistry

The apparatus, systems and methods disclosed herein can also be used inthe study of photochemical fractionations that occur in the Earth'satmosphere. Indeed, intra-molecular isotopic distributions have manyuses, such as, for example, the study of atomistic reaction mechanisms(e.g., the study of the Diels-Alder reaction of isoprene and maleicanhydride to form cyclohexene products), diffusion, gravitationalsettling, mixing, high-dimensionality “fingerprinting,” deconvolvingcomplex budgets, and chemical or pyrolitic degradation (e.g.,ozonolysis, oxidation, and/or decarboxylation; such as the degradationof palmitoleic acid).

The above-described applications of the apparatus, systems and methodsdisclosed herein are technically straightforward when applied tocompounds that are intrinsically volatile and are readily available athigh concentrations (e.g., have high abundances in the blood stream,such as glucose or urea).

Embodiments of the second mass spectrometer will now be described inmore detail. The aspects, features and methods described with respect tothe second mass spectrometer may also be available for embodiments ofthe first mass spectrometer described herein. According to an embodimentof the invention, the second mass spectrometer has the ionizationcapabilities of a gas source isotope ratio mass spectrometer and themass resolution, sensitivity and versatility of analyzers and detectorarrays of ion microprobe and inductively coupled plasma massspectrometers. An embodiment of the second mass spectrometer is shown inFIG. 27. The spectrometer shown in FIG. 27 may be a normal geometry,double-focusing sector mass spectrometer. A sector mass spectrometer ofthis kind is suitable as an embodiment of the “second massspectrometer,” because such instruments are capable of multi-collection,permitting concurrent (or simultaneous) analysis of ion beam intensitiesat two or more cardinal masses. In the embodiment shown in FIG. 27, thesecond mass spectrometer 10 includes a second ion travel path along asecond entrance slit 39, a second energy filter 40 (e.g., anelectrostatic analyzer or “ESA”) and a second momentum filter 60 (e.g.,a magnetic sector) configured to provide ions (e.g., sixth molecularanalyte ions) to a detector array 80. The second mass spectrometer canbe configured to provide a second mass resolution (which is described inmore detail below) of 20,000 or greater at the detector array bysequentially arranging the second entrance slit, the second energyfilter and the second momentum filter, and by appropriately selecting asecond width of the second entrance slit, a third radius of curvature ofthe second energy filter and a fourth radius of curvature of the secondmomentum filter.

The second mass resolution achieved by a second mass spectrometeraccording to embodiments of the invention (e.g., a magnetic sector massspectrometer) is generally proportional to the separation distancebetween two ion beams that the second mass spectrometer can achieve forion beams that include respective ions having masses that are differentfrom one another. The separation distance between the ion beams isproportional to the fourth radius of curvature of the second momentumfilter (i.e., magnet) along the second ion travel path, and inverselyproportional to the width of each ion beam, which is proportional to thewidth of the second entrance slit. Additionally, the highest massresolutions can be achieved by momentum filtering (e.g., magnetic sectormass spectrometry) if the ions being filtered by momentum havesubstantially uniform kinetic energy. Thus, according to embodiments ofthe invention, the ions are filtered by energy (e.g., by the energyfilter) prior to being filtered by momentum (e.g., by the momentumfilter). Accordingly, the second energy filter has dimensions that areconsistent with the creation of a double-focusing condition at the iondetector, given the accelerating potential of the ions as they exit thesecond ion source and the radius of the second momentum filter. Forexample, second mass resolutions in the range of about 2,000 to about20,000 can be achieved if the second entrance slit has a second width ofabout 250 μm to about 5 μm, respectively, the ions are accelerated to 5keV after exiting the second ion source, the third radius of curvatureof the energy filter along the second ion travel path is about 20 cm toabout 25 cm, the ions are further accelerated by an additional 5 keVafter the second energy filter, and the fourth radius of curvature ofthe second momentum filter (e.g., magnetic sector) is about 20 cm toabout 25 cm. In one embodiment, the second width of the second entranceslit is about 5 μm, the third radius of curvature of the second energyfilter is about 22 cm, and the fourth radius of curvature of the secondmomentum filter is about 23 cm.

Embodiments of the second mass spectrometer also include a source of ananalyte and a source of a reference material. For example, as shown inFIG. 27, the second mass spectrometer 10 can also include a sampleintroduction apparatus 20 (e.g., a second sample introductionapparatus), which is configured to provide an analyte gas (or othergases) to a second ion source 30. The second ion source is configured toconvert the analyte gas (or other gases, such as various referencematerials described below) to ions (e.g., fourth molecular analyteions). The ion source produces the ions as a first output (e.g., thefourth molecular analyte ions). As the ions exit the ion source, theyencounter the second entrance slit 39, which can be included as acomponent of the second ion source or can be connected to the second ionsource. The second entrance slit is configured to guide the first outputof molecular analyte ions (the fourth molecular analyte ions) along thesecond ion travel path.

The second energy filter 40 (e.g., the ESA) is positioned along thesecond ion travel path downstream from the second entrance slit 39 andis configured to receive the first output of molecular analyte ions (thefourth molecular analyte ions), which have energy levels. The secondenergy filter has a third radius of curvature along the second iontravel path and is configured to filter out fifth molecular analyte ionsfrom the fourth molecular analyte ions according to their energy levelsand produce a second output of molecular analyte ions. The second energyfilter can be any suitable device that can filter ions according totheir energy levels, such as an ESA.

A first ion focusing element 50 can be included along the second iontravel path between the second energy filter 40 and the second momentumfilter 60. The first ion focusing element is configured to focus thesecond output of molecular analyte ions (the fifth molecular analyteions) along the second ion travel path to the second momentum filter.The first focusing element can be any suitable device capable offocusing the second output of molecular analyte ions (the fifthmolecular analyte ions), such as an electrostatic or magnetic lens(e.g., a quadrupole or higher format lens).

The second momentum filter 60 is positioned along the second ion travelpath downstream from the second ion source 30, the second entrance slit39, the second energy filter 40 and the first ion focusing element 50,and is configured to receive the second output of molecular analyte ions(the fifth molecular analyte ions). The second momentum filter has afourth radius of curvature along the second ion travel path and isconfigured to filter out sixth molecular analyte ions from the fifthmolecular analyte ions according to their momenta and produce a thirdoutput of molecular analyte ions. The second momentum filter can be anysuitable device that can filter ions according to their momenta, such asa magnetic sector.

A second ion focusing element 70 can be included along the second iontravel path between the second momentum filter 60 and the detector array80. The second ion focusing element is configured to focus the thirdoutput of molecular analyte ions (the sixth molecular analyte ions)along the second ion travel path to the detector array. The second ionfocusing element can be any suitable device capable of focusing thethird output of molecular analyte ions (the sixth molecular analyteions), such as an electrostatic or magnetic lens (e.g., a quadrupole orhigher format lens).

The detector array 80 is positioned downstream of the second momentumfilter 60 (and the second ion focusing element 70) and is configured toreceive the third output of molecular analyte ions (the sixth molecularanalyte ions). The detector array may be any suitable device orcombination of devices capable of concurrently detecting two or moremolecular analyte ions of the third output of molecular analyte ions(the sixth molecular analyte ions), of which the two or more molecularanalyte ions have masses that are different from one another (and massto charge ratios that are different from one another).

The second mass spectrometer 10 can be configured to provide the thirdoutput of the molecular analyte ions (the sixth molecular analyte ions)to the detector array 80 at a second mass resolution (which is describedin more detail below) of 20,000 or greater. For example, the width ofthe second entrance slit 39 and the third and fourth radii of curvatureof the second energy filter and second momentum filter can be selectedto provide a second mass resolution at the detector array of 20,000 orgreater. In one embodiment, the third output of molecular analyte ions(the sixth molecular analyte ions) includes at least two ion beams andrespective molecular analyte ions of the ion beams have respectivemasses that differ from one another by about 1 atomic mass unit, and thesecond width, third and fourth radii of curvature, and detector arrayare configured to resolve and concurrently detect the at least two ionbeams and to distinguish between molecular analyte ions within each ionbeam at one part in 20,000. The detector array is configured toconcurrently detect the at least two molecular analyte ions. Thedetector array can be connected to a processor (or processors) 90 (e.g.,a computer or computers), which can be configured to acquire data fromthe detector array and to process the data. As described in more detailbelow, the processor (e.g., computer) can also be configured to controlvarious features of the second mass spectrometer, such as the detectorarray. In some embodiments, some components of the second massspectrometer 10 are controlled by processors separate from and inaddition to the processor 90.

Embodiments of the individual components of the second mass spectrometerwill now be described in more detail. FIG. 28 shows an embodiment of thesample introduction system 20 coupled to the second ion source 30through five separate conduits (e.g., capillaries). The sampleintroduction system of FIG. 28 includes a system of valves andreservoirs suitable for alternate introduction of samples and referencestandards into a mass spectrometer ion source. Any of the followingconduits can be heated to facilitate the delivery of an analyte orreference material to the ion source. The sample introduction system isconfigured to provide analyte(s) or reference material(s) to the ionsource as a neutral gas. The analyte(s) or reference material(s) can beintroduced to the second ion source as a pure gas through a viscouscapillary bleed (e.g., a flow of gas through a capillary column) or theycan be entrained (or mixed) in a flow (e.g., a continuous flow or apulsed flow) of any suitable carrier gas (e.g., an inert gas, such ashelium gas).

The sample introduction system can selectively introduce the analyte orthe reference material to the ion source. For example, in the embodimentshown in FIG. 28, the sample introduction system includes anintroduction tube 8 configured to receive pure sample gases 7, such asanalyte or reference material gases. A first valve 1 (e.g., a firsthigh-vacuum pneumatic valve) is configured to control the flow of thegases into the introduction tube. The introduction tube is furthercoupled to four sets of valves, bellows and conduits (e.g., capillaries)arranged in parallel and coupled to a valve block configured toselectively couple the introduction tube to the ion source through eachset of valves, bellows and conduits.

As shown in FIG. 28, the introduction tube 8 is coupled to a secondvalve 2, a third valve 3, a fourth valve 4, a fifth valve 5 and a sixthvalve 6, each of which can be a high-vacuum pneumatic valve. The secondthrough fifth valves are each configured to selectively couple theintroduction tube to a first bellows 11, a second bellows 12, a thirdbellows 13, and a fourth bellows 14, respectively. The first throughfourth bellows 11-14 are each coupled to a first conduit 15, a secondconduit 16, a third conduit 17, a fourth conduit 18, respectively. Thefirst through fourth conduits 15-18 (e.g., the first through fourthcapillaries) are coupled to a valve block 25 (e.g., a change-over valveblock), which is coupled to the second ion source 30. The sixth valve iscoupled to a high-vacuum system 9.

By appropriately selecting the first through sixth valves 1-6, a gas,such as an analyte, analytes or reference material, can be introducedinto the introduction tube and confined therein. For example, the firstvalve 1 can be opened to introduce a gas to the introduction tube, andone of the second through fifth valves 2-5 can be selected to couple theintroduction tube to one of the first through fourth bellows 11-14,respectively, to thereby provide the gas to the selected bellows. Byopening the first valve, a gas can be introduced into the introductiontube, and by opening the second valve 2, the gas can be provided to thefirst bellows 11. From the first bellows, the gas can then be providedto the first conduit 15 (e.g., the first capillary), which is coupled tothe valve block 25. The valve block can selectively couple the firstconduit to the second ion source 30. Thus, the sample introductionsystem 20 can provide a gas to the second ion source through the firstvalve, the introduction tube, first bellows, first conduit andchange-over block. Similarly, other gases can be provided to the secondion source through the first valve, introduction tube, third throughfifth valves, second through fourth bellows, second through fourthconduits and valve block. The sixth valve is configured to selectivelycouple the introduction tube to the high-vacuum (HV) system 9 toevacuate and purge the introduction tube when switching between gases.As shown in FIG. 28, the valve block is also selectively coupled to anHV system 19 configured to evacuate and purge the valve block whenswitching between gases. The HV systems 9 and 19 can be the same ordifferent. Any of the above-described introduction tube, first throughsixth valves, first through fourth bellows and valve block can be heatedto facilitate delivery of an analyte or reference material to the ionsource.

In the embodiment shown in FIG. 28, the sample introduction system 20further includes a gas chromatograph 22 coupled to the second ion source30 through a fifth conduit 23 (e.g., a fifth capillary) including anopen split 24. A sample can be injected into the gas chromatographthrough an injector 21 and the sample can be separated by the gaschromatograph to provide a purified analyte (e.g., a specific compoundor specific set of compounds) to the fifth conduit, which transmits theanalyte through the open split to the second ion source. Any suitablegas chromatograph can be used. The analyte can be entrained in a carriergas flow (e.g., He) that flows through the gas chromatograph, the fifthconduit and the open split to the second ion source. The open split isconfigured to allow the sample introduction system to provide equivalentconditions to the second ion source regardless of whether an analyte isprovided by the chromatograph to the fifth conduit. The fifth conduitand/or open split can be heated to facilitate delivery of an analyte orreference material to the second ion source.

The above-described first through fifth conduits 15-18 and 23 areconfigured to provide two or more streams of matter (e.g., analyte(s) orreference material(s)) to the second ion source 30. The conduits can beconfigured to accommodate: (1) a sample of purified compounds that aregases at room temperature; (2) a capillary bleed of carrier gas (e.g.,He delivered through the open split 24 and fifth conduit 23); and (3-5)three separate reference materials (e.g., reference gases) that differfrom one another in their isotopic compositions by known amounts. Withrespect to (2), the capillary bleed of carrier gas can be configured toserve as the carrier gas for volatile organic compounds, such asvolatile organic compounds introduced through the gas chromatograph 22.Each of the conduits (e.g., capillaries) described above is capable ofintroducing analyte(s) or reference material(s) to the second ion sourceas a stream of matter and of being separately and independentlyselected.

As discussed in more detail below, in some embodiments, measurements ofan analyte (or analytes) are standardized to concurrently analyzedstandards (e.g., reference materials) and, therefore, the sampleintroduction system 20 can be configured to deliver the analyte (oranalytes) to the second ion source 30 through two or more separateconduits described above. Using the sample introduction system,reference materials can be preselected such that inter-comparison of thereference materials can be used to determine the mass discrimination ofthe second mass spectrometer source, the reaction constants for relevantfragmentation/adduct reactions at the second ion source, and thelinearity of the second mass spectrometer detector system.

The sample introduction system 20 can be any apparatus that is suitablefor use with a gas source isotope ratio mass spectrometer, such as, forexample, a mercury bellows, an automated mechanical bellows (e.g., thedual inlet systems of the mass spectrometers available from NuPerspective or the MAT-253 mass spectrometer available from ThermoFisher Scientific, Inc., Waltham, Mass.), a He-purged carrier gas systemthat interfaces with a capillary bleed through an open split, or thelike. Any apparatus capable of delivering the analyte with flowsufficient to support pressures at the second ion source 30 on the orderof 10⁻⁶ mbar can be used. Backing pressures of several mbar to about 1bar are generally achieved using a capillary bleed having an interiordiameter of tens to hundreds of microns. In some embodiments, the secondmass spectrometer 10 includes a modified version of the “front end” ofthe MAT-253 mass spectrometer available from Thermo Fisher Scientific,Inc., including a sample introduction system including 4 bellows and acarrier-gas port (as described above with respect to FIG. 28), and amodified second ion source 30 as shown in FIG. 29. The sampleintroduction system and second ion source can be modified to have thecharacteristics described herein and to be compatible with the othercomponents of the second mass spectrometer described herein. Forexample, in one embodiment, the sample introduction system 20 includeseach of the components of the sample introduction system of the MAT-253mass spectrometer available from Thermo Fisher Scientific, Inc., withthe components of the sample introduction system physically rearrangedto fit within a cabinet that differs in size and shape from that of theMAT-253.

In the embodiment shown in FIG. 29, the second ion source includes anionization chamber 35 between a trap 32 (e.g., an anode) and a filament34 (e.g., a hot cathode). The ion source can further includeelectrostatic lenses and apertures generally similar to those used inother gas source mass spectrometers. For example, in FIG. 29, the secondion source further includes an extraction lens 36, a shield 37, andfocusing/grounding elements 38. Neutral gas 31 (e.g., analyte orreference material gas) enters the ionization chamber and molecular ionsare generated by electron impact. The molecular ions are then extractedas an ion beam 33, accelerated and focused by the extraction lens,shield, and focusing/grounding elements. The second ion source canprovide molecular ions with an initial acceleration of about 5 kV. InFIG. 29, the second entrance slit 39 is adjacent to the second ionsource, and the ion beam is further focused or narrowed as it exits thesecond ion source through the second entrance slit.

The second ion source 30 can be any suitable ion source, such as thoseincluding an electron-impact ionization chamber resembling the Nier-typeion source used in existing gas-source isotope ratio mass spectrometers.For example, the second ion source can be a Nier-type ion sourceavailable from Nu Perspective or Thermo Fisher Scientific, Inc. (e.g.,the ion source of the MAT-253 mass spectrometer) that has been modifiedby expanding the range of electron impact energy to extend down to atleast 5 eV, rather than the standard lower limit of 50 eV. In oneembodiment, the second ion source 30 is the ion source of the MAT-253mass spectrometer available from Thermo Fisher Scientific, Inc.,machined to fit within a housing that differs in size and shape fromthat of the MAT-253, and machined to fit together with the othercomponents of the second mass spectrometer.

An ion source capable of providing an electron impact energy of lessthan 50 eV provides improved control over the fragmentation spectrum ofthe molecular ions as compared to an ion source that has a 50 eV lowerlimit on electron impact energy. The second ion source can be configuredto have a voltage potential between the source filament (e.g., thefilament 34) and the housing of the ionization chamber (e.g., the trap32) that is adjustable in a range of at least 5 eV to less than or equalto 150 eV. For example, the second ion source can be capable ofproviding an electron impact energy of less than 50 eV, such as an ionsource that is configured to provide an electron impact energy of about5 eV to about 150 eV, or about 25 eV to about 150 eV.

As shown in FIG. 29, the second entrance slit 39 is the last apertureencountered by the ion beam 33 as it exits the second ion source 30. Thesecond entrance slit can be adjacent to the second ion source, and itcan be between the second ion source and the detector array 80. In someembodiments, the second entrance slit has a variable aperture. Forexample, the second entrance slit can be adjustable to a second width ina range of about 10 μm to about 250 μm, such as a second width of 5 μmto about 250μ, or a second width of about 5 μm. The second entrance slitcan be adjustable, either continuously or through movement of two ormore fixed-width apertures (having the same or different fixed-widths),such that the ion beam width can reach the intended mass resolution ofabout 20,000 or greater at the detector array. For example, the secondentrance slit width can be achieved by using two or more slits movablerelative to one another to achieve the desired width. Thus, the secondwidth of the second entrance slit can vary between 5 μm and about 250 μmby way of a mechanical device that translates slits of variable width inand out of the path of the ion beam through the second mass spectrometeranalyzer. In some embodiments, when the second mass spectrometer hasoverall dimensions and ion optics similar to those of conventionalhigh-resolution inductively coupled mass spectrometers, an entrance slitas small as 5 to 10 μm can be used.

Referring back to FIG. 27, the second energy filter 40 is configured toreceive the first output of molecular analyte ions (the fourth molecularanalyte ions) from the second entrance slit 39. The second energy filtercan be any suitable device capable of separating ions according to theirenergy levels, such as an electrostatic analyzer. The second energyfilter can be dynamically pumped to maintain the interior of the secondenergy filter under high-vacuum. The second energy filter can have athird radius of curvature of about 20 cm to about 25 cm, which may beequal (or roughly equal) to the fourth radius of curvature of the secondmomentum filter, provided that the kinetic energy of the ions enteringthe second energy filter is about one half of the kinetic energy thatthe ions will have when entering the second momentum filter. Forexample, a third radius of curvature of the second energy filter ofabout 20 cm to about 25 cm will provide suitable mass resolution at thedetectors if the ions are accelerated to one half of their final energyprior to entering the second energy filter and the ions are thenaccelerated to their full final energy after energy filtering. Forexample, a third radius of curvature can be about 22 cm. One or moreelectrostatic lenses may be used to shape, focus and/or center the ionbeam before the ion beam enters the second energy filter. Similarly, oneor more electrostatic lenses may be used to shape, focus and/or centerthe ion beam between the second energy filter and the second momentumfilter, and/or between the second momentum filter and the detectors. Thesecond energy filter can provide the first output of molecular analyteions (the fourth molecular analyte ions) with about 5 kV of accelerationin addition to the acceleration provided by the second ion source 30. Asan example, the second energy filter can be the energy filter of aNEPTUNE mass spectrometer or TRITON mass spectrometer (each of which areavailable from Thermo Fisher Scientific, Inc.), modified to have theabove-described characteristics and to be compatible with the othercomponents of the second mass spectrometer described herein. Forexample, in one embodiment, the second energy filter is theelectrostatic analyzer of the NEPTUNE mass spectrometer available fromThermo Fisher Scientific, Inc.

In FIG. 27, the second energy filter 40 is configured to produce asecond output of molecular analyte ions (the fifth molecular analyteions). The second output can pass through the first ion focusing element50, which can function as a transfer lens (e.g., a quadrupole or higherformat lens). The first ion focusing element can be configured to focusthe second output of molecular ions. For example, in one embodiment, thefirst ion focusing element is one of the ion focusing elements of theNEPTUNE mass spectrometer available from Thermo Fisher Scientific, Inc.

The second momentum filter 60 is positioned downstream from the secondenergy filter 40 and the first ion focusing element 50, and isconfigured to receive the second output of molecular analyte ions (thefifth molecular analyte ions). The second momentum filter has a fourthradius of curvature and is configured to filter out sixth molecularanalyte ions from the fifth molecular analyte ions according to theirmomenta. The second momentum filter produces a third output of molecularanalyte ions (the sixth molecular analyte ions). The second momentumfilter can have a fourth radius of curvature of about 20 cm to about 25cm. For example, the second momentum filter can include a magnet havinga fourth radius of curvature of about 23 cm. As an example, the secondmomentum filter can be the momentum filter of a NEPTUNE massspectrometer or TRITON mass spectrometer (each of which are availablefrom Thermo Fisher Scientific, Inc.), modified to have theabove-described characteristics and to be compatible with the othercomponents of the second mass spectrometer described herein. Forexample, in one embodiment, the second momentum filter is the magneticsector of the NEPTUNE mass spectrometer available from Thermo FisherScientific, Inc.

A second ion focusing element 70 can be positioned downstream of thesecond momentum filter 60. The second ion focusing element can beconfigured to focus the third output of molecular analyte ions (thesixth molecular analyte ions). For example, the second ion focusingelement can be a “zoom” lens, such as a dispersion quadrupole or higherformat lens. In some embodiments, the second ion focusing element has“zoom” optic capability (±5% mass range) and is configured to provide 2×magnification at the image plane of the detector array 80. For example,in one embodiment, the second ion focusing element is one of the ionfocusing elements of the NEPTUNE mass spectrometer available from ThermoFisher Scientific, Inc.

As shown in FIG. 27, the detector array 80 is positioned downstream ofthe second momentum filter 60 and the second ion focusing element 70. Atleast a portion of the molecular ions (e.g., analyte ions, referencematerial ions, etc.) that pass through the second momentum filter andsecond ion focusing element are detected at the detector array. Forexample, the detector array can be configured to receive the thirdoutput of molecular analyte ions (the sixth molecular analyte ions) fromthe second momentum filter.

The detector array 80 can be a multi-collector array including two ormore detectors (e.g., 5 to 10 detectors) capable of faraday-cup or ioncounting detection at each of several positions. For example, FIG. 30 isa partial schematic view of the detector array 80 including detectors82. As shown in FIG. 30, the ion current of the ion beams 81, 83 and 85arriving at the respective detectors is registered through either anelectron multiplier 84 (or ion counting system having similarperformance characteristics) or faraday cup 86 current monitoring systemto enable quantitative analysis over a large dynamic range insensitivity at each mass position. The ion beams can include molecularanalyte ions or reference material ions, and the ions of the respectiveion beams have masses that are different from one another. For example,the third output of molecular analyte ions (the sixth molecular analyteions) can include the ion beams 81, 83 and 85.

Both the position and sensitivity (e.g., ion counting vs. faraday cupcurrent measurement) of at least one detector 82 can be controlled in anautomated fashion (e.g., through the computer 90 of FIG. 27) so that thecollection characteristics of the detector can be adjusted on timescales of minutes, without disturbing the delivery of analyte to thesecond mass spectrometer source or the vacuum within the second massspectrometer. The foregoing switching capability is beneficial, becausemost analyses of interest benefit from comparison of measured ratios fortwo or more molecular or fragment ions derived from the same analytespecies (see examples, below).

In some embodiments, the detector array 80 can be reconfigured over thecourse of measurements made on a single sample (e.g., a single analyte),and the reconfiguration can be both rapid and convenient. At least oneof the detectors 82 is capable of movement relative to the otherdetectors such that the relative positions of detected ions can beadjusted. For example, in FIG. 30, the detectors can vary their relativespacing as shown by the arrows 87. The detectors can vary their positionalong the focal or image plane of the second mass spectrometer, which isindicated by the dashed line 88. The detectors can be sufficientlymobile to permit rapid reconfiguration to achieve a wide range ofrelative and absolute mass positions, up to a mass to charge ratio ofabout 300. Reconfiguration of detector position can be motorized andautomated, and can be performed through stepper motors or analogousmechanical devices, which can be controlled and powered remotely throughvacuum feed-throughs.

Switching between ion counting and current monitoring detectors at eachdetector position can be achieved through electrostatic deflectors atthe exit slit positioned before each detector position. The arrows 89indicate the electrostatic deflection that can be used to switch betweendetection by ion counting (e.g., detection by the electron multiplier84) and detection by current measurement (e.g., detection by the faradaycup 86).

In one embodiment, the detector array 80 includes seven detectors 82,six of which are movable. Each detector includes a faraday cup (FC) andan electron multiplier (EM), and each detector is switchable between FCand EM measurement. Additionally, signals can be collected from each FCand EM concurrently (or simultaneously). Detector signals are convertedto digital measures of intensity (e.g., ion current or counts-per-secondrates) using digital-to-analog (DAC) circuits common to the detectorsystems of several commercially available isotope ratio massspectrometers, and then delivered to the computer 90 (shown in FIG. 27)capable of storing relative ion beam intensities for later dataprocessing. In one embodiment, the detector array further includes aretarding potential quadrupole (RPQ) lens upstream of the centraldetector, such that ions pass through the RPQ lens prior to arriving atthe central detector.

Examples of suitable detector arrays include the detector arrays of theCameca ims 1280 microprobe and NanoSIMS ion probes, each available fromCameca, Société par Actions Simplifiée, and the detector array of theTRITON thermal ionization mass spectrometer, available from ThermoFisher Scientific, Inc.

According to embodiments of the invention, the above-describedcomponents can be arranged to provide a double-focusing, normal-geometrysector mass spectrometer having an ion beam size, mass separation andsystem stability sufficient to achieve a mass resolution at the detectorarray of 20,000 or greater (mass/ΔM, according to the 5%-95% definition,which is described in more detail below). The second mass spectrometercan have the following capabilities: a vacuum under analyticalconditions of not more than 10⁻⁸ mbars; useful ion yield of not lessthan 1 ion per 5×10⁴ molecules at the highest mass resolution; a massrange of about 2 to about 300 atomic mass units (“AMU”), such as a massrange of about 2 to about 280 AMU; a mass resolution on the order of20,000 (according to the mass/ΔM, 5%-95% definition, which is describedin more detail below); and abundance sensitivity of not more than 10⁻⁶.Other mass spectrometers are not capable of multi-collection ofmolecular analyte ions (e.g., concurrently detecting two or moremolecular analyte ions, the molecular analyte ions having differentmasses) generated by electron impact ionization at a mass resolution of20,000 or greater (as described in more detail below). Embodiments ofthe invention further include a Nier-type gas source and associatedinlet system to the analyzer, such that high-resolution multi-collectormass spectrometry can be performed on molecular ions generated byelectron impact on gases and volatile compounds (e.g., volatile organiccompounds). The second mass spectrometer has a mass range sufficient foranalyzing ions of large volatile organic molecules (e.g., phytane) andtheir associated fragments. When the second mass spectrometer has a massresolution at the detector array of 20,000 or greater, the second massspectrometer can resolve isobaric interferences among isotopologues oforganic molecules (e.g., internal isobars, such as isotopologues havingthe same cardinal mass), their fragments, their adducts, and contaminantspecies (e.g., contaminants having the same cardinal mass as themolecular analyte ion or molecular analyte fragment ion being measured).These features are not found in other mass spectrometer designs that arealso capable of multiple simultaneous collection of two or more ionbeams.

Methods

Embodiments of the invention are also directed to methods fordetermining the isotopic composition of a compound (e.g., an analyte),such as methods of using the above-described second mass spectrometer10. For example, FIG. 31 is a flow chart illustrating a method fordetermining the isotopic composition of an analyte in a sample. Themethod includes (100) converting an analyte to molecular analyte ions.The analyte can be converted to the molecular analyte ions using asecond ion source of a second mass spectrometer (e.g., the second ionsource 30 described above). The ion source can produce the molecularanalyte ions from the analyte. The method further includes (110)separating at least a portion of the molecular analyte ions using asecond entrance slit to produce a first output of molecular analyte ions(fourth molecular analyte ions). The molecular analyte ions from the ionsource can be separated using the above-described second entrance slit39 to produce the first output (the fourth molecular analyte ions). Asdescribed above, the second entrance slit 39 can have an adjustablesecond width. The second width can be adjusted to vary the separation ofthe molecular analyte ions from the second ion source and to adjust thesecond mass resolution of the spectrometer at the detector array (e.g.,the above-described detector array 80).

The method of FIG. 31 also includes (120) further separating at least aportion of the molecular analyte ions of the first output (the fourthmolecular analyte ions) according to their energy levels to produce asecond output (e.g., filtering out fifth molecular analyte ions from thefourth molecular analyte ions according to their energy levels). Thefurther separating of the molecular analyte ions of the first output canbe accomplished using the above-described second energy filter 40 (e.g.,the electrostatic analyzer). The degree of the further separation of themolecular analyte ions by the second energy filter depends upon thethird radius of curvature of the second energy filter. Thus, asdescribed above, the third radius of curvature of the second energyfilter affects the second mass resolution of the spectrometer 10 at thedetector array 80. The second energy filter can have any of theabove-described third radii of curvature. The second energy filterfilters out the fifth molecular analyte ions from the fourth molecularanalyte ions according to their energy levels.

The method further includes (130) separating at least a portion of themolecular analyte ions of the second output according to their momentato produce a third output (e.g., filtering out sixth molecular analyteions from the fifth molecular analyte ions according to their momenta).The above-described second momentum filter 60 (e.g., the magneticsector) can be used to separate the molecular analyte ions of the secondoutput according to their momenta. The degree of separation of themolecular analyte ions by the second momentum filter depends upon thefourth radius of curvature of the second momentum filter. Thus, asdescribed above, the fourth radius of curvature of the second momentumfilter affects the mass resolution of the spectrometer 10 at thedetector array 80. The second momentum filter can have any of theabove-described fourth radii of curvature. The second momentum filterfilters out the above-described third molecular analyte ions from thesecond molecular analyte ions according to their momenta.

Multi-Collection/Detection

The method shown in FIG. 31 further includes (140) concurrentlydetecting (e.g., multi-collection) two or more molecular analyte ions ofthe third output (the sixth molecular analyte ions) to produce secondmolecular analyte ion data, the two or more molecular analyte ionshaving respective masses that are different from one another (andrespective mass to charge ratios that are different from one another).As shown in FIG. 31, there are two different approaches tomulti-collection.

In the first approach (140 a), the two or more molecular analyte ions ofthe third output (the sixth molecular analyte ions) are detected usingtwo or more detectors to produce the second molecular analyte data. Thisembodiment is referred to as “parking,” since each of the molecular ionbeams is “parked” at one detector. According to this embodiment, theanalyte may be introduced to the second ion source 30 by the sampleintroduction system 20 as a continuous flow or as a time resolved pulse.Concurrent detection by parking is suitable for detecting molecular ionshaving respective masses that differ by at least 1 AMU. Molecular ionsthat differ by less than 1 AMU may not be sufficiently resolved to beconcurrently detected at separate detectors.

According to this embodiment, the two or more molecular ions (e.g.,molecular analyte ions) differ from one another by at least 1 AMU andcan be concurrently (or simultaneously or quasi-simultaneously) detectedin two separate detectors. FIG. 32 shows an example of concurrentdetection by “parking” in which a first molecular ion beam 81 isdetected at a first detector 82 a and a second molecular ion beam 83 isconcurrently detected at a second detector 82 b. The intensity ratio ofthese two separately registered (e.g., detected) signals at any one timeis the measure of the abundance ratio of the two relevant isotopicspecies (e.g., the respective molecular ions). The intensities detected(or registered) for the first ion beam 81 and second ion beam 83 areeach recorded and averaged over a specified period of time (generallyseconds). In FIG. 32, the first detector 82 a and the second detector 82b are part of the same detector array 80.

This method of concurrent (or simultaneous or quasi-simultaneous)detection of ions by “parking” can be employed when the intensities ofthe ion beams exiting the second ion source do not vary substantiallyover time, for example when delivering a stable (e.g., continuous) flowof gas to the ion source through a capillary bleed, or when theintensities of the ion beams vary through time, for example when analyteis delivered to the second ion source as a brief pulse in a heliumcarrier gas stream. This method also further includes (150 a) analyzingthe second molecular analyte ion data to determine the isotopiccomposition of at least a portion of the analyte. Other ions (e.g.,reference material ions) can also be analyzed in the second massspectrometer in a similar manner.

In the second approach (140 b), the two or more molecular analyte ionsare detected by scanning the sixth molecular analyte ions (or ion beamsincluding the respective molecular analyte ions) across at least onedetector. In some embodiments, scanning at least one molecular analyteion beam across at least one detector produces a change in a detectedsignal intensity as masses of molecular analyte ions detected by thedetector change at an amount of one part in 20,000. This method isreferred to as “peak scanning,” since the ion beams are scanned across adetector. This method of scanning can be employed when the analyte isdelivered to the second ion source 30 from the sample introductionsystem 20 as a continuous flow (e.g., through a capillary bleed thatvaries little in flow rate over time). This method of scanning the ionbeams across a single detector is unsuitable, however, for analyses ofbrief pulses of analyte, such as those delivered to the second ionsource as components of a helium carrier gas.

FIGS. 33A-D illustrate the scanning of a first molecular ion beam 81 anda second molecular ion beam 83 across a single detector 82 of a detectorarray 80. FIG. 33E illustrates the mass spectrum that results from thescanning of the ion beams across the single detector in FIGS. 33A-D. Asshown in FIG. 33A, the first molecular ion beam enters the detectorfirst. In the mass spectrum of FIG. 33E, the first molecular ion beam isshown entering the detector at 91. Then, as shown in FIG. 33B, thesecond molecular ion beam enters the detector, resulting in both ionbeams being detected concurrently (or simultaneously) in the detector.The second molecular ion beam is shown entering the detector at 93 ofthe mass spectrum of FIG. 33E, at which point the measured signal is acomposite signal that includes a contribution from each of the first andsecond molecular ion beams. Then, the first molecular ion beam exits thedetector as shown in FIG. 33C. At this point only the second molecularion beam is detected in the detector. The first molecular ion beam isshown leaving the detector at 95 of the mass spectrum of FIG. 33E. Then,as shown in FIG. 33D, the second molecular ion beam exits the detector.The second molecular ion beam is shown leaving the detector at 97 of themass spectrum of FIG. 33E.

FIG. 33F illustrates the portions of a measured signal intensity thatcan be used to calculate the first mass resolution of the first massspectrometer at the detector or the second mass resolution of the secondmass spectrometer at the detector array. In FIG. 33F, 90% (5%-95%) ofthe measured signal intensity for a particular mass is contained in thewidth ΔM between the two vertical arrows. The mass resolution can becalculated by dividing the mass of the ion measured by the width ΔM.Accordingly, as used herein, the term “mass resolution” refers to thevalue calculated by dividing the mass of the ion measured by the widthΔM that contains 90% of the measured signal intensity.

The above-described scanning results in a time-varying detected ion beamintensity in the detector across which the ion beams are scanned, withan example of a resultant mass spectrum shown in FIG. 33E. The scanningcan take place over a time period of seconds or minutes. The ion beamscan be scanned by adjusting the accelerating potential of the ion beams(e.g., by adjusting the accelerating potential of the ion source 30 orthe energy filter 40) or by adjusting the magnetic field strength of thesecond momentum filter 60 (e.g., by adjusting the intensity of a currentdelivered to an electromagnet included in the second momentum filter).

The peak scanning method of detecting ions can be employed when the ionbeams include respective ions that have similar, but not identical, massto charge ratios (e.g., the respective ions each have the same cardinalmass but are at least partially discriminated from one another in thesecond mass spectrometer analyzer). Accordingly, the peak scanningapproach can be applied when the sixth molecular analyte ions haverespective masses that differ from one another by less than 1 AMU. Thepeak scanning approach can also be applied when the sixth molecularanalyte ions have respective masses that differ from one another by atleast 1 AMU (or more than 1 AMU). This method also further includes (150b) analyzing the molecular analyte ion data to determine the isotopiccomposition of at least a portion of the analyte. Other ions (e.g.,reference material ions) can be analyzed in the second mass spectrometerin a similar manner. While the above-described “parking” approach and“peak-scanning” approach can be carried out separately, measurementsmade using parking and peak-scanning can be used in a single analysis.

The above-described analyzing, as well as that described below, can becarried out as described in the above referenced U.S. ProvisionalApplication No. 61/652,095, filed on May 25, 2012, the entire contentsof which are incorporated herein by reference. The analyzing can alsoutilize one or more databases of isotopic information. The databases canbe generated using the methods and apparatus described herein, or theycan be generated using numerical simulations. A person of skill in theart would recognize the type of isotopic information that should beincluded in a database to be used in the methods described herein. Forexample, such databases would include commonly observed proportions offragment and adduct ions in the full mass spectrum of analytes ofinterest, as measured under common (or consistent) instrumental tuningconditions (including properties such as the electron impact energy ofthe ion source and source pressure of the analyte or compound beingintroduced into the second mass spectrometer).

Standardization

All analyses described herein can be standardized by comparison withreference materials (e.g., reference analytes) having known (or preset)isotopic compositions, including predetermined (or preset) proportions(or concentrations) of isotopologues of interest. The referencematerials can be analyzed under conditions (i.e., chemical purity,ion-source pressure and instrument settings) that are closely similar tothose of the unknown samples (e.g., the analyte). Additionally, thereference materials can be converted to ions, separated and detectedaccording the methods described above with respect to the analyte. Thedescription provided below illustrates some means by which thesestandards can be created and characterized. As described below, analysisof the standards (e.g., reference materials) can be used to calibrateseveral instrumental artifacts.

For example, standardization can include alternate measurement of asample (e.g., an analyte) and a standard (e.g., one or more referencematerials) according to the methods described herein (e.g., convertingthe analyte or standard to ions, separating and detecting the ions asdescribed above with respect to the analyte). In one embodiment, each ofthe analyte and the standard is drawn from a relatively large (˜10⁻⁶ molor larger) reservoir of gas (e.g., a gas containing the analyte orstandard) and delivered to the second ion source through a capillarybleed (e.g., one of the conduits 15-18 described with respect to FIG.28) at a rate that varies little with time (e.g., as a continuous flow).For example, the standard can be drawn from the relatively largereservoir of gas by the sample introduction system and introduced intothe second ion source. The standard can then be converted to ions by thesecond ion source and the ions can be separated by the second entranceslit, the second energy filter and the second momentum filter. Theseparated ions of the standard can be detected by the detectors and thenanalyzed. The process can then be repeated for analyte drawn fromanother relatively large reservoir of gas that is different from thestandard. The process can also then be repeated for another standardthat is the same as or different from the first standard. Each time thegas stream entering the second ion source (e.g., the second ion source30) is alternated from sample to standard or standard to sample, theoperator can wait several seconds until ion intensities reach stable,relatively time-invariant values before recording signal intensities.

As with the analyte, reference materials delivered by the sampleintroduction system 20 as a continuous flow can be detected by eitherconcurrently detecting the reference material ions using two or moredetectors (e.g., the above-described concurrent detection 140 a or“parking”) or by scanning the reference material ion across at least onedetector (e.g., the above-described concurrent detection 140 b or “peakscanning”).

When the reference material ions are concurrently detected by two ormore detectors (e.g., the above-described concurrent detection 140 a or“parking”), intensities detected (or registered) for two or moreseparate masses are recorded and averaged over a specified period oftime (generally seconds) before switching the gas flow to anotherreservoir (e.g., from sample to standard or standard to sample). Thisprocess is repeated two or more times, generating a time series ofobservations of two or more ion intensities (and thus one or moreintensity ratios) for sample (e.g., analyte) and standard (e.g.,reference material). Interpolation between any two standard measurementsprovides the basis for standardizing the intervening sample measurement.Aspects of this method are based on techniques common to existingdual-inlet gas source isotope ratio mass spectrometers.

When the reference material ions are concurrently detected according tothe “peak scanning” approach 140 b, all reference ion beams having asingle cardinal mass are scanned across at least one detector 82 (e.g.,at least one collector). The reference ion beams can be scanned byadjusting the accelerating potential of the ion beams (e.g., byadjusting the accelerating potential of the second ion source 30 or thesecond energy filter 40) or by adjusting the magnetic field strength ofthe second momentum filter 60 (e.g., by adjusting the intensity of acurrent delivered to an electromagnet included in the second momentumfilter).

As with the analyte, the “peak scanning” can be done using a singledetector, or using two or more detectors as part of the same scan. Thisresults in a time varying intensity at each detector, where variationsin intensity reflect changing proportions of the various ion speciesthat contribute to the population of ions at each cardinal mass, asshown in the mass spectrum of FIG. 33E. According to this embodiment,gas flow to the second ion source should be relatively stable over thetime scale of each scan (though subtle variations may be corrected forby introducing a modest correction to intensity as a function of time toaccount for depletion of a vapor reservoir being analyzed, or othersimilar artifacts). Resulting composite peaks can be de-convolved forthe relative intensities of their component ion beams by methods readilyunderstood by those of skill in the art, such as through an algorithmthat assumes an initial guess as to the number, identity and relativeintensities of the component ion beams and then iteratively solves forthe least-squares best fit relative intensities of those ion beams.

For example, a program, such as a MATLAB® script (MATLAB is a registeredtrademark of The Mathworks Inc., Delaware USA), can be used to constructa synthetic data set by stipulating, for example, the shapes of the ionbeams (e.g., the widths of the ion beams), the width of the detector,the intensity of a first ion beam, and the intensity of a second ionbeam. The program can then produce a simulated mass spectrum based onthe stipulated conditions and compare the simulated mass spectrum to themeasured mass spectrum. The program can then iteratively solve for theleast-squares best fit relative intensities of the ion beams (e.g., bysearching for the set of conditions that best match the measured massspectrum) to thereby determine the relative contribution of each ionbeam to the measured mass spectrum. Standardization can be achieved byperforming the above-described operations for a standard gas stream(e.g., analyte or reference material of the same chemical compositionand source pressure as the sample but having a known or presetcomposition) near in time to the analysis of the sample (e.g., theanalyte), and under closely or relatively similar source pressures andinstrument settings to those used for the sample. The relativeintensities of component ion beams determined for sample (e.g., analyte)and standard (e.g., reference material) are recorded and used in one ormore of the standardization schemes described below.

In an alternative embodiment, a sample (e.g., an analyte) and a standard(e.g., a reference material) can be measured alternately, where each isintroduced to the second ion source (e.g., the second ion source 30)through time-resolved pulses contained within a helium carrier gasstream that continuously flows into the second ion source or is added tothe second ion source through a separate capillary bleed concurrent withintroduction of the helium carrier gas. According to this embodiment,the method includes concurrently detecting (e.g., concurrently detectingaccording to the “parking” 140 a described above), for any one pulse ofsample (standard or analyte), the ion intensity of molecular ions havingtwo or more different masses for any one pulse of sample, standard oranalyte, the masses of the molecular ions differing from one another byat least 1 AMU. The ion intensities are integrated over the duration ofthe pulse. Signals registered during periods of unstable or negligiblysmall signal intensities at the beginning and end of each pulse can beomitted to improve the quality of the data obtained. Standardization canbe achieved by comparing ion intensity ratios measured for pulses of theanalyte of the sample to those measured for pulses of the standard(e.g., the reference material) introduced before and/or after the sampleanalyte pulse, either by the averaging of all bracketing standardanalyses or by interpolation between any two bracketing standardanalyses. Aspects of this method are based on that common to existingcarrier gas isotope ratio mass spectrometers.

Examples of several parameters (e.g., instrumental artifacts) that canbe standardized are described in more detail below.

Instrumental Mass Bias

The mass discrimination in the above-described second ion source 30,second energy filter 40 and detector array 80 of the second massspectrometer 10 can be calibrated by comparing mass discriminationreference data (e.g., an ion intensity (I_(i)) ratio) for two or moremass discrimination reference ion beams to the known (or preset)concentrations of the isotopologues or isotopomers from which the massdiscrimination reference ion beams are produced by electron impactionization. For example, as shown in FIG. 34, calibrating the massdiscrimination of the second mass spectrometer can include (300)obtaining mass discrimination reference data from a mass discriminationreference material including mass discrimination reference isotopologuesor isotopomers A-MD and B-MD that differ in their respective mass tocharge ratios and have respective mass discrimination referenceconcentrations [A-MD] and [B-MD]. The mass discrimination reference datacan be obtained by analyzing the mass discrimination reference materialaccording to the methods described above with respect to the analyte.The mass discrimination reference data includes mass discriminationreference ion intensities I_(A-MD) and I_(B-MD) corresponding to therespective mass discrimination reference isotopologues or isotopomersA-MD and B-MD. The method according to this embodiment can furtherinclude (310) determining the mass discrimination of the second massspectrometer by comparing a ratio of the mass discrimination referenceion intensities I_(A-MD) and I_(B-MD) to a ratio of the massdiscrimination reference concentrations [A-MD] and [B-MD] using aconstant of proportionality α_(IMF) according to the Equation:I _(A-MD) /I _(B-MD)=([A-MD]/[B-MD])α_(IMF)

The molecular analyte ion data acquired according to the methodsdescribed above can then be modified using the constant ofproportionality α_(IMF) (320). The isotopic composition of at least aportion of the analyte can then be determined from the modifiedmolecular analyte ion data.

Instrument Linearity

The instrument “linearity” (L) can be defined as a constant ofproportionality between a measured ratio of reference ion intensityratios for two reference materials (e.g., standards) to a ratio ofabundance ratios of the relevant parent isotopologues or isotopomers.For example, as shown in FIG. 35, calibrating the linearity of thesecond mass spectrometer 10 can include (400 a) obtaining firstlinearity reference data from a first linearity reference materialincluding first linearity reference isotopologues or isotopomers A-1 andB-1 at a first linearity reference concentration ratio ([A-1]/[B-1])₁.The first linearity reference data can be obtained by analyzing thefirst linearity reference material according to the methods describedabove with respect to the analyte. The first linearity reference dataincludes a first linearity reference intensity ratio (I_(A-1)/I_(B-1)) ₁corresponding to the first linearity reference isotopologues orisotopomers A-1 and B-1. The method further includes (400 b) obtainingsecond linearity reference data from a second linearity referencematerial including second linearity reference isotopologues orisotopomers A-2 and B-2 at a second linearity reference concentrationratio ([A-2]/[B-2])₂. The second linearity reference data can beobtained by analyzing the second linearity reference material accordingto the methods described above with respect to the analyte. The secondlinearity reference data includes a second linearity reference intensityratio (I_(A-2)/I_(B-2))₂ corresponding to the second linearity referenceisotopologues or isotopomers A-2 and B-2 (400 b). The method furtherincludes (410) determining the linearity (L) of the second massspectrometer according to the Equation:(I _(A-1) /I _(B-1))₁/(I _(A-2) /I_(B-2))₂={([A-1]/[B-1])₁/([A-2]/[B-2])₂ }L

The method can further include modifying the molecular analyte ion datausing the linearity L (420). The isotopic composition of at least aportion of the analyte can then be determined from the modifiedmolecular analyte ion data (430). The linearity of the second massspectrometer is an empirically measured analytical artifact and isexpected to be specific to each instrument, analytical condition (e.g.,analyte type, source pressure and instrument tuning condition) andmeasured ratio of interest. Thus, it can be calibrated by comparison oftwo or more standards (e.g., reference materials) that differ by a known(or preset) amount in abundance ratios of isotopic species of interest.

Fragmentation Probability

Some embodiments of the invention include measurements of ions that arecharged fragments of analyte molecules. As a result, it can be useful tocalibrate a relationship between an ion intensity of a fragment ofinterest (e.g., a molecular analyte fragment ion intensity or I_(FA)) toan intensity of corresponding to an ion of the intact molecule fromwhich it is derived (e.g., an intact molecular analyte ion intensity orI_(A-molecular)). This can be achieved by calibration of a constant ofproportionality K_(fragmentation) for the fragmentation reaction throughanalysis of the intensity ratio of the molecular analyte fragment ion tothe intact molecular analyte ion. For example, the molecular analyteions can include the intact molecular analyte ions and the molecularanalyte fragment ions. Each of the molecular analyte ions are formed byionizing an intact molecule of the analyte and each of the molecularanalyte fragment ions are formed by dissociating one or more of theintact molecules of the analyte or the intact molecular analyte ions.Additionally, the molecular analyte ion data includes the molecular ionintensity I_(A-molecular) corresponding to one or more of the intactmolecular analyte ions. The molecular analyte ion data also includes themolecular analyte fragment ion intensity I_(FA) corresponding to one ormore of the molecular analyte fragment ions.

As shown in FIG. 36, embodiments of the invention include (500)determining a ratio of the molecular analyte fragment ions to the intactmolecular analyte ions by calculating a constant of proportionalityK_(fragment) according to the Equation:I _(FA) /I _(A-molecular) =K _(fragment)

The method also includes modifying the molecular analyte ion data usingthe constant of proportionality K_(fragment) (510). The isotopiccomposition of at least a portion of the analyte can then be determinedfrom the modified molecular analyte ion data (520). The constant ofproportionality K_(fragment) is an empirically measured analyticalartifact, and is expected to be specific to each instrument, analyticalcondition (e.g., analyte type, source pressure and instrument tuningcondition) and measured ratio of interest.

Adduct Probability

Some embodiments of the invention include measurements of ions that areions or ionic fragments of the analyte molecules which have gained oneor more excess H (or, potentially, other) atoms. As a result, it can beuseful to calibrate a relationship between an ion intensity of adductions of interest (e.g., an analyte adduct ion intensity orI_(A′-adduct)) to an intensity corresponding to an ion of theun-adducted molecule from which it is derived (e.g., the molecularanalyte ion intensity or I_(A-molecular)). This can be achieved bycalibration of a constant of proportionality K_(adduct) for theadduction reaction through analysis of the intensity ratio of themolecular analyte adduct ion to the intact molecular analyte ion. Forexample, the molecular analyte ions can include intact molecular analyteions and analyte adduct ions. Each of the intact molecular analyte ionsare formed by ionizing an intact molecule of the analyte and each of themolecular analyte adduct ions are formed by combining one or more of theintact molecules of the analyte or the analyte ions and a hydrogen atomor an other material, the other material being the same as or differentfrom the analyte molecules or the analyte ions. Additionally, themolecular analyte ion data includes an intact molecular analyte ionintensity I_(A-molecular) corresponding to one or more of the intactmolecular analyte ions and a molecular analyte adduct ion intensityI_(A′-adduct) corresponding to one or more of the molecular analyteadduct ions.

As shown in FIG. 37, embodiments of the invention include (600)determining a ratio of the molecular analyte adduct ions to the intactmolecular analyte ions by calculating a constant of proportionalityK_(adduct) according to the Equation:I _(A′-adduct) /I _(A-molecular) =K _(adduct)

The method further includes modifying the molecular analyte ion datausing the constant of proportionality K_(adduct) (610). The isotopiccomposition of at least a portion of the analyte can then be determinedfrom the modified molecular analyte ion data (620). The constant ofproportionality K_(adduct) is an empirically measured analyticalartifact, and is expected to be specific to each instrument, analyticalcondition (e.g., analyte type, source pressure and instrument tuningcondition) and measured ratio of interest. The constant ofproportionality K_(adduct) is also expected to vary with the partialpressure of water in the ion source, and/or abundances in the ion sourceof other potential sources of H other than the analyte molecules.

Redistribution or Recombination Probability

Molecular ions can be generated by chains of electron-molecule,electron-ion, ion-ion and ion-molecule reactions that result infragmentation of a parent species (e.g., an intact analyte molecule) andrecombination of the resultant fragments to re-form ions that areindistinguishable in mass from the parent molecule (e.g., the intactanalyte molecule or an ion thereof). Such reactions are sufficientlyenergetic that they effectively drive the sample (e.g., the analyte)toward a random distribution of isotopes among all possibleisotopologues. Such effects, sometimes referred to as “scrambling,”commonly result in redistribution of isotopes among isotopologues ofapproximately several percent, relative, of all measured molecular ions(e.g., molecular analyte ions). The foregoing effects can bestandardized by comparison of two or more standards (e.g., referencematerials) that differ by known (or preset) amounts in their isotopicdistributions. In most instances, these effects cannot be standardizedthrough analysis of a single ion beam intensity or ion intensity ratio;rather, one must monitor the change from expected values of theequilibrium constant for an isotope exchange reaction involving ahomogeneous reaction among isotopologues of the same molecule.

For example, as shown in FIG. 38, the method according to embodiments ofthe invention can include (700 a) obtaining first recombinationreference data from a first recombination reference material includingfirst recombination reference isotopologues or isotopomers A-1 and B-1and having a preset first recombination reference ratio of the firstrecombination reference isotopologues or isotopomers A-1 to the firstrecombination reference isotopologues or isotopomers B-1. The firstrecombination reference data can be obtained by analyzing the firstrecombination reference material according to the methods describedabove with respect to the analyte. The method also includes (710 a)determining a constant of proportionality K_(eq) ¹ from the firstrecombination reference data. The method further includes (700 b)obtaining second recombination reference data from a secondrecombination reference material including second recombinationreference isotopologues or isotopomers A-2 and B-2 and having a presetsecond recombination reference ratio of the second recombinationreference isotopologues or isotopomers A-2 to the second recombinationreference isotopologues or isotopomers B-2. The second recombinationreference data can be obtained by analyzing the second recombinationreference material according to the methods described above with respectto the analyte. The method also further includes (710 b) determining aconstant of proportionality K_(eq) ² from the second recombinationreference data, and (720) determining a rate of recombination of ions,molecules and electrons in the second mass spectrometer according to theEquation:(K _(eq) ¹ /K _(eq) ²)_(measured)/(K _(eq) ¹ /K _(eq) ²)_(known) =K_(redistribution)

The method also includes modifying the molecular analyte ion data usingthe constant of proportionality K_(redistribution) (730). The isotopiccomposition of at least a portion of the analyte can then be determinedfrom the modified molecular analyte ion data (740).

As an example of recombination and redistribution, K_(eq) can be definedto be the equilibrium constant for the following reaction among methaneisotopologues:¹³CH₄+¹²CH₃D↔¹³CH₃D+¹²CH₄

For example, K_(eq)=[¹³CH₃D][¹²CH₄]/[¹³CH₄][¹²CH₃D], and K_(eq) can bedetermined as the product of two isotopologue ratios (e.g.,[¹³CH₃D]/[¹²CH₃D]x[¹²CH₄]/[¹³CH₄]). As set forth above, a correctionfactor for redistribution through “scrambling” (e.g.,K_(redistribution)) can be calculated through comparison of values ofK_(eq) for two known standards (e.g., reference materials), K_(eq) ¹ andK_(eg) ². FIG. 39 is a graph illustrating the temperature dependence ofthe reaction ¹³CH₄+¹²CH₃D↔¹³CH₃D+¹²CH₄. As can be seen in FIG. 39,¹³CH₃D enrichment decreases as the temperature increases.

Migration Probability

Electron impact ionization is capable of driving migration of atomsbetween positions of an ion (e.g., an intact molecular analyte ion, ananalyte fragment ion or a molecular analyte adduct ion). For example,the term “migration” can be used to describe the process by which ahydrogen atom in a methyl (CH₃) group of a propane molecule tradespositions with a hydrogen atom in the CH₂ position of that samemolecule. For example, FIG. 40 is a graph illustrating the temperaturedependence of such a migration in the equilibration reaction:¹²CH₂D-¹²CH₂-¹²CH₃↔¹²CH₃-¹²CHD-¹²CH₃. As can be seen in FIG. 40, Denrichment at the C2 position of propane decreases as the temperatureincreases. Such an effect can be standardized by comparison of twostandards (or reference materials) that differ from one another by aknown (or preset) amount in the concentrations of an isotope of interestin the two sites being investigated. For example, hydrogen isotopeexchange between the two sites (‘1’ and ‘2’) of a propane moleculethrough hydrogen migration can be calibrated according to the equation:([D] ₁ /[D] ₂)_(Measured) =[D] ₁ /[D] ₂)_(Known) ×K _(migration)

As shown in FIG. 41, according to embodiments of the invention themethod can include (800) obtaining migration reference data from amigration reference material including isotopomers having an initialmigration reference concentration ratio ([D]₁/[D]₂)_(initial). Themigration reference data can be obtained by analyzing the migrationreference material according to the methods described above with respectto the analyte. The method also includes (810) determining a measuredmigration reference concentration ratio ([D]₁/[D]₂)_(measured) from themigration reference data. The method further includes (820) determininga constant of migration K_(migration) according to the Equation:([D] ₁ /[D] ₂)_(measured)/([D] ₁ /[D] ₂)_(initial) =K _(migration)

The molecular analyte ion data can be modified using the constant ofproportionality K_(migration) (830). The isotopic composition of atleast a portion of the analyte can then be determined from the modifiedmolecular analyte ion data (840).

The above-described reference materials (e.g., the mass discriminationreference material, the first linearity reference material, the secondlinearity reference material, the first recombination referencematerial, the second recombination reference material, and the migrationreference material) may each be any suitable material (e.g., anysuitable compound) and they may each have a composition that is the sameor different as that of the other reference materials or the analyte.

Standardization

In some embodiments, standardization of any or all of theabove-described phenomena for unknown samples (e.g., unknown analytes)is independently or concurrently accomplished through “sample-standardbracketing,” as described above, such as sequential analysis of astandard (e.g., a reference material), a sample of unknown composition,and a standard (e.g., another reference material that is the same as ordifferent from the first reference material). For example, referencedata can be first be obtained by analyzing a reference materialaccording to the methods described above with respect to the analyte.Next analyte data can be obtained by analyzing an analyte data accordingto the methods described above. Then, additional reference data can beobtained by analyzing a reference material (which can be the same as ordifferent from the reference material above) according to the methodsdescribed above with respect to the analyte. After the reference data,analyte data and additional reference data has been obtained, linearinterpolation of the values measured for at least one or more of theconstants α_(IMF), L, K_(fragment), K_(adduct), K_(redistribute), orK_(migration) (determined from the reference data and additionalreference data according to the methods described above) for thebracketing standards to determine the values of the constants thatshould be applied to the unknown sample.

For example, as shown in FIG. 42, the method according to embodiments ofthe invention can include (900) converting the analyte to molecularanalyte ions using an ion source in a second mass spectrometer. Themethod also includes (910) separating at least a portion of themolecular analyte ions by mass to charge ratio to provide two or moremolecular analyte ion beams. The method further includes (920) obtainingmolecular analyte ion data from the two or more molecular analyte ionbeams. The molecular analyte ion data can be obtained according to themethods described above. The method also further includes (1000)obtaining one or more of mass discrimination reference data, linearityreference data, recombination reference data, and migration referencedata, and (1010) determining one or more of a linearity (L), a constantof proportionality α_(IMF), a constant of proportionality K_(adduct), aconstant of proportionality K_(fragment), a constant of proportionalityK_(redistribution), and a constant of migration K_(migration), which canbe determined according to the methods described above. The methodfurther includes modifying the molecular analyte ion data using one ormore of α_(IMF), L, K_(adduct), K_(fragment), K_(redistribution), andK_(migration) (1100). The isotopic composition of at least a portion ofthe analyte can then be determined from the modified molecular analyteion data (1110).

The number and frequency of the above-described bracketing measurementscan vary with instrument conditions, analyte type and desired accuracyand precision. Based on prior experience with broadly similarinstrumentation and measurements, α_(IMF) can be calibrated on timescales of tens of seconds (e.g., using the dual inlet changeover valveor multiple-injection carrier gas methods common to existing gas sourceisotope ratio mass spectrometers), values of K_(fragment) and K_(adduct)will be relatively constant over periods of hours to days, and values ofL and K_(redistribute) will be constant over periods of days to weeks.There is insufficient prior evidence to predict the variability ofK_(migration). The design purpose of having 4 gas reservoirs, each withits own capillary bleed, in the sample introduction system 20 (e.g., theinlet system) is to facilitate convenient comparison of an unknownsample (e.g., a sample including an analyte such as a gas or volatileorganic compound introduced through a helium carrier gas) with multiplestandards of known isotopic composition.

Statistical Determinations of Isotope Distributions

The methods described above are capable of determining the abundanceratios of isotopologues of molecular species and their derivatives(e.g., fragments thereof, adducts thereof, etc.). However, additionalcalculations can be used to establish positions of isotopes within thesemolecular species based on comparisons of respective isotopiccompositions of a molecule and one or more of its fragments, or two ormore of its fragments. This can be achieved through a combination ofprinciples of sampling statistics with standardization of relevantanalytical constants (e.g., α_(IMF), L, K_(fragment), K_(adduct),K_(redistribute), K_(migration)), as described above.

For example, propane (C₃H₈) contains two non-equivalent carbonpositions: a central (CH₂) position (hereafter, position ‘A’) and twosymmetrically equivalent terminal methyl (CH₃) positions (hereafterposition ‘M’). The difference in ¹³C concentration between these twopositions can be determined by comparison of the abundance ratios:[¹³C¹²C₂H₈]/[¹²C₃H₈] and [¹³CH¹²CH₅]/[¹²C₂H₅], through simultaneoussolution of the following family of equations (i.e., equations 1 through4):[¹³C¹²C₂H₈]/[¹²C₃H₈]=(2[¹³C]_(M)[¹²C]_(M)[¹²C]_(A)+[¹³C]_(A)[12C]_(M)²)/([¹²C]_(M) ²[¹²C]_(A)])  (1)[¹³C¹²CH₅]/[¹²C₂H₅]=([¹³C]_(M)[¹²C]_(A)+[¹³C]_(A)[12C]_(M))/([¹²C]_(M)[¹²C]_(A)])  (2)[¹³C]_(M)+[¹²C]_(M)=1  (3)[¹³C]_(A)+[¹²C]_(A)=1  (4)

In equations 1 and 2, [¹³C]_(M), [¹³C]_(A), [¹²C]_(M) and [¹²C]_(A)refer to the ¹³C and ¹²C concentrations of the methyl (M) and alkyl (A)molecular sites.

Two additional expressions must hold true to account for the fullisotopic inventory of both methyl and alkyl sites (i.e., [¹³C]_(M),[¹³C]_(A), [12C]_(M), and [¹²C]_(A) must satisfy equations 3 and 4).

There are four unknowns ([¹³C]_(M), [¹³C]_(A), [¹²C]_(M) and [¹²C]_(A))in the above family of four equations. Thus, one can uniquely solve thisfamily of equations, assuming that [¹³C¹²C₂H₈]/[¹²C₃H₈] and[¹³C¹²CH₅]/[¹²C₂H₅] have been correctly determined through massspectrometric measurements of the relevant ion ratios (i.e.,standardized to determine the relevant analytical constants) andassuming that the C₂H₅ fragment derives from the loss of one methylgroup of the parent molecule (this last assumption is consistent withprior study of the fragmentation physics of propane, and can be verifiedon one embodiment of the second mass spectrometer of the presentinvention through analyses of standards that have been highly enrichedin ¹³C in the central and terminal carbon positions). Thus, it isstraightforward to convert two measurable ratios of molecular orfragment ions into fully constrained determinations of the proportionsof ¹³C contained in two structurally distinct molecular sites. Similarreasoning can be applied to a large number of other instances in whichisotopic contents of positions in organic and other molecular structurescan be reconstructed from the analysis of molecular and fragment ions.

Ion Correction of Non-Resolved Isobaric Interferences

Some molecular and fragment ion species may be difficult to resolve fromnearby isobaric interferences, either due to the high total mass of thespecies in question or because the species is so low in abundance thatit is preferable to perform mass spectrometric analysis with a largeentrance slit and correspondingly high transmission but poor massresolution. In such cases, abundances of unresolved species of interestmay be determined by correction based on independent constraints on theproportions of all species contributing to a composite ion peak. ¹²CH₃Dmethane serves as an illustrative example. The abundance of ¹²CH₃D couldbe determined without direct mass resolved analysis if one measured theion intensity ratio: (I_(13CH4)+I_(12CH3D)+I_(12CH5))/I_(12CH4) andsubtracted contributions from the two interfering species (¹³CH₄ and¹²CH₅) by separately measuring ¹³CH₄/¹²CH₄ (a relatively easily measuredratio at moderate mass resolution) and K_(adduct) (which can bedetermined through analyses of standard gases or reference materials, asdescribed above).

EXAMPLES

Three examples of uses of the apparatus and methods according toembodiments of the invention follow. The examples are supported withdata generated on a second mass spectrometer according to one embodimentof the invention. The second mass spectrometer used in the examplesdemonstrated a mass resolution (e.g., resolving power) up to 25,500using the above described 5%-95% definition of mass resolution. Table 1shows a comparison of the sensitivity and resolving power (e.g., massresolution) of a second mass spectrometer according to exemplaryembodiments of the invention to a conventional MAT-253 mass spectrometeravailable from Thermo Fisher Scientific, Inc.

TABLE 1 Entrance Resolving Power slit (μm) Molecules/ion (5%/95%)Conventional MAT-253 500 600 ~500 Example 1 250 ~1,500 1,500-2,000 50~7,500  7,500-10,000 25 ~15,000 10,000-12,000 15 ~20,000 12,000-15,000 5~60,000 20,000-25,000

As can be seen in Table 1, the second mass spectrometer according toembodiments of the invention exhibit significantly higher resolvingpower, but reduced sensitivity as compared to the conventional MAT-253mass spectrometer available from Thermo Fisher Scientific, Inc. However,most stable isotope ratio measurements are made at ion currents>10⁹ CPSand, therefore, the relatively lower sensitivity of embodiments of theinvention is acceptable, as one can sacrifice a lot of signal and stillobtain a precise measurement.

The second mass spectrometer according to embodiments of the inventionalso demonstrated flat plateaus on hydrocarbon “ziggurat” peaks (e.g.,peaks having the shape of a truncated pyramid, such as a pyramid havinga flat top) as shown in the mass spectrum of FIG. 33E. The second massspectrometer according to embodiments of the invention has alsodemonstrated ˜0.1‰ precision on faraday cup/electron multiplier ratiomeasurements. The second mass spectrometer according to embodiments ofthe invention is noteworthy for its flexibility, mass range, ease of usefor complex spectra, and it has significantly improved resolving powerrelative to ICP-MS instruments, which could be the result of: thestability of the Nier-type electron impact (EI) source, the narrowenergy distribution of the source, and the improved vacuum inembodiments of the invention.

The apparatus, systems and methods according to embodiments of theinvention described herein can be used in various applications. Forexample, according to embodiments of the invention, a method ofidentifying a high-potential oil-field includes analyzing an analyte ofa sample from a target field using an embodiment of the second massspectrometer and/or methods described herein to obtain relativeproportions of isotopologues in one or more samples, such as methane,ethane or propane. The relative proportions of the isotopologues can beused to calculate equilibrium constants for isotope exchange reactionsamong the isotopologues (such as the reaction among methaneisotopologues described below). Temperatures of gas formation or storagecan be inferred by comparing these calculated equilibrium constants tothe temperature-dependent values calibrated by theory or experiment.These temperatures can then be compared with known geothermal gradientsto infer the depths in the earth of hydrocarbon generation and/orstorage, which can then be used to guide drilling for oil and gasexploration.

Example 1: Methane Thermometry

As described above, the temperature of origin of methane or otherhydrocarbons is useful for natural gas exploration and study of theenvironmental chemistry of methane. For example, the temperature oforigin of methane constrains the depths and mechanisms of the sourcerocks from which the methane was obtained, and the temperature ofstorage informs exploration of the reservoirs where the methane istrapped. The temperatures of equilibration of molecules such as methanecan be recorded by the proportions of their rare, heavy isotopes (e.g.¹³C and D) that form multiply substituted isotopologues (e.g., ¹³CH₃D)rather than singly substituted isotopologues (e.g., ¹³CH₄ and ¹²CH₃D).The proportions of the rare, heavy isotopes of methane are relatedthrough exchange reactions, such as:¹²CH₃D+¹³CH₄↔¹³CH₃D+¹²CH₄

The equilibrium constants for the exchange reactions of methane, such asthe one shown above, are a function of temperature, and thusmeasurements of relative proportions of the four isotopologues involvedin the reaction above provides a method of geochemical thermometry(e.g., a method of determining the temperature of origin and/or storageof a sample of methane). Such methods of thermometry have beenpreviously determined for CO₂ and carbonate, and the principles ofthermodynamics that explain this phenomenon are well known of H₂, O₂,N₂, CO, N₂O and a variety of other simple molecular compounds. Similarprinciples apply to the isotopic abundances of multiply substitutedisotopologues of ethane (e.g., ¹³C₂H₆) and higher order hydrocarbons.

The principles of statistical thermodynamics and the spectroscopy ofmethane indicate that the equilibrium constant for the above exchangereaction is ˜1.005 at 300 K and decreases toward 1 roughly linearly withdecreasing value of the quantity (1/T²), as shown in FIG. 43. FIG. 43includes predicted equilibrium constants for isotope exchange reactionsinvolving homogeneous equilibria of methane isotopologues, includingmultiply substituted isotopologues. The calculations were based ondensity functional theory predictions of the vibrational dynamics ofmethane and its isotopologues and quantum mechanical models of therelevant partition functions. Calculated equilibrium constants werenormalized by the value for a random distribution of isotopes among allisotopologues and plotted as per mil (‰) deviations from that randomdistribution. A sufficiently precise and accurate measurement of therelative abundances of the four isotopologues of methane appearing inthe above equation should permit one to determine the temperature offormation of methane. Similar principles lead to predictions regarding alarge number of similar homogeneous isotope exchange equilibria amongisotopologues of alkanes and other organic and inorganic molecules.

Table 2 below provides the precise masses and expected abundances of thefour relevant isotopologues of methane for a sample equilibrated at 300K and in which 1% of its carbon atoms are ¹³C and 0.015% of its hydrogenatoms are D (i.e., approximately the natural isotopic abundances). Thethird and fourth columns of Table 2 list the expected (or predicted) ioncurrents, in both amps and counts per second, for all analyzed speciesat the detector, assuming an ion source with performance characteristicssimilar to commercial Nier-type electron impact sources (having typicalionization efficiencies), a gas pressure in the source similar to theoperating conditions of common stable isotope ratio mass spectrometers(e.g., ˜10⁻⁷ mbar), and 1% transmission (e.g., a reduction intransmission of a factor of 100, corresponding to the transmission lossassociated with use of the smallest source aperture to achieve high massresolution). A usefully precise temperature estimate requires that thesmallest of these ion intensities be measured with precision of ˜0.01%.

TABLE 2 Mass Ion current Counts per Species (AMU) Concentration (amps)second# ¹²CH₃D 17.0376 5.94E−04 1.80E−12 1.13E+07 ¹³CH₄ 17.0347 9.99E−033.03E−11 1.89E+08 ¹³CH₃D 18.0409 6.00E−06 1.82E−14 1.14E+05 ¹²CH₄16.0313 9.89E−01 3.00E−09 1.88E+10 #Values in excess of 10⁶ are too highfor analysis by electron multiplier; these ion beams are analyzed byfaraday cup collection.

As can be seen in FIG. 44A, which shows a mass spectrum (logarithmicscale) acquired for methane, the second mass spectrometer according toan embodiment of the invention is capable of mass-resolving tworepresentative isotopologues relevant to this measurement (¹²CH₄ fromthe ion fragment, ¹³CH₃; it is not visually obvious, but both speciesare also discriminated from the minor beam of ¹²CH₂D in this spectrum).The peak shape shown in FIG. 44A was generated by scanning the mass 16AMU ion beam of methane across a single detector, using a second massspectrometer according to one embodiment of the invention. The locationsat which the ¹³CH₃ ⁺, ¹²CH₂D⁺ and ¹²CH₄ ⁺ ion beams enter and then exitthe detector are shown in the mass spectrum of FIG. 44A.

Additionally, FIGS. 44B-G show the ¹³CH₃ ⁺, ¹²CH₂D⁺ and ¹²CH₄ ⁺ ionbeams entering and then exiting a single detector 82 of a detector array80. For example, FIG. 44B shows the ¹³CH₃ ⁺ ion beam entering thedetector. FIG. 44C shows the ¹²CH₂D⁺ ion beam entering the detector suchthat both the ¹³CH₃ ⁺ and ¹²CH₂D⁺ ion beams are detected in the singledetector concurrently (or simultaneously). Next, FIG. 44D shows the¹²CH₄ ⁺ ion beam entering the detector such that all three of the ¹³CH₃⁺, ¹²CH₂D⁺ and ¹²CH₄ ⁺ ion beams are detected in the single detectorconcurrently (or simultaneously). FIG. 44E shows the ¹³CH₃ ⁺ ion beamexiting the detector such that the ¹²CH₂D⁺ and ¹²CH₄ ⁺ ion beams aredetected in the single detector concurrently (or simultaneously). FIG.44F shows the ¹²CH₂D⁺ ion beam exiting the detector such that only the¹²CH₄ ⁺ ion beam is detected in the detector. FIG. 44G shows the ¹²CH₄ ⁺ion beam exiting the detector.

The mass resolution of the peak scan shown in FIG. 44A is about 25,500(using the 5%-95% definition described above). As can be seen in FIG.44A, the second mass spectrometer according one embodiment of theinvention is capable of resolving ¹³CH₃ from ¹²CH₄. While it is notimmediately clear in FIG. 44A, the minor ion beam ¹²CH₂D is alsowell-resolved from ¹³CH₃ from ¹²CH₄. Similar performance is expected fordiverse ions of methane, its fragments and adducts having othermass/charge ratios.

FIG. 45 demonstrates the external precision that can be achieved throughsample/standard bracketing in measurements of the ratio:(¹³CH₄+¹²CH₃D+¹²CH₅)/¹²CH₄ (i.e., the mass 17/16 ratio) for methane.These measurements were taken using a second mass spectrometer accordingto one embodiment of the invention. Each point represents the standarddeviation (1 s) of measured ratios, where each measurement is theaverage over a 1 second or 115 second integration. Measurements weremade at two different source pressures, to vary the counting rate. Allmeasurements were made using electron multiplier detectors. Thehorizontal axis is the predicted standard error of each suchmeasurement, based on counting statistics. As can be seen in FIG. 45,the above-described ratio was measured with external precision as goodas 0.01 percent, relative, over a wide range of current integrationtimes and signal intensities, indicating that the second massspectrometer according to an embodiment of the invention is capable ofmeasuring ion intensity ratios with precision sufficient for the exampleapplications described herein.

The mass resolutions and the precision of the above-described isotoperatio measurements are sufficient such that embodiments of the inventioncan be used to calibrate values of α_(IMF) and L for the two isotoperatios measured (e.g., [¹³CH₃D]/[¹²CH₃D] and [¹²CH₄]/[¹³CH₄]) and forK_(redistribution) for methane in the ion source. Three standards (e.g.,reference materials) having distinct, known (or preset) isotopiccompositions can be used to calibrate all three of these analyticalconstants prior to analysis of an unknown sample (e.g., a samplecontaining methane as an analyte having unknown isotopic composition).This standardization can be accomplished using embodiments of theinvention through the repeated sequential analysis of three standards(e.g., reference materials) and one sample of unknown composition (e.g.,an analyte having unknown isotopic composition), by alternatelymeasuring the gas streams emanating from four flexible bellows of theinlet system of the second mass spectrometer. K_(fragmentation) andK_(migration) are not relevant to any of the species analyzed for theabove-described analysis of methane; if required to make measurementsfor analogous applications to molecules more complex than methane,K_(fragmentation) and K_(migration) can be calibrated through study ofadditional standards.

Measurements similar to those above may also be useful for carbondioxide (CO₂), which has 18 naturally occurring isotopologues (¹²C¹⁶O₂,¹²C¹⁸O₂, ¹⁴C¹⁸O¹⁷O, etc.), each of which is unique in its physical andchemical properties and thus constitutes a potential independent tracerof source, reaction mechanism and/or environment of origin. Othermethods of mass spectrometric or spectroscopic analysis of CO₂isotopologues are capable of determining relative abundances of only 5of these species (¹²C¹⁶O₂, ¹³C¹⁶O₂, ¹⁴C¹⁶O₂, ¹²C¹⁸O¹⁶O, and ¹²C¹⁷O¹⁶O).Any information encoded in the proportions of the other remaining 13species is effectively lost by those other measurements.

Example 2: Position-Specific Isotope Composition of n-Alkanes

Another embodiment is directed to the determination of relativeabundances of ¹³C-bearing isotopologues of the CH₃ ⁺ and C₂H₅ ⁺ ionfragments generated by ionization of propane. The foregoing data,combined with characterizations of the empirical constants describingfragmentation and recombination reactions in the ion source, can be usedto determine the difference in ¹³C content between the terminal andcentral carbon positions of propane. This difference is predicted to bea function of temperature in thermodynamically equilibrated propane (andthus can be used to establish the temperature of formation, as for themethane analysis described above). In non-equilibrated gases, thisdifference may illuminate the chemical kinetic mechanisms of natural gasmaturation, and thus also aid in the exploration and development of oiland gas deposits.

Naturally occurring n-alkanes (e.g., methane, ethane, propane, etc.) areproducts of diverse processes, such as thermal degradation of organicmatter, hydrothermal reactions of aqueous solutions, and biosynthesis;many are also products of industrial chemical synthesis. The carbon andhydrogen isotope content of the n-alkanes is a function of both thecarbon sources from which they were synthesized and the conditions andchemical mechanisms of their synthesis. Thus, forensic identificationand source attribution of organic molecules may be achieved based on theisotopic fingerprints of the organic molecules, including but notrestricted to the carbon and hydrogen isotope compositions of n-alkanes.Measurements made with embodiments of the invention include severalnovel constraints to the isotopic fingerprint of alkanes and therebyfacilitate determination of the sources of the alkanes. In embodimentsof the invention, some of the novel measurements include: determinationof the ¹³C/¹²C ratios of non-equivalent sites in alkane structures,determination of the D/H ratios of non-equivalent sites in alkanestructures, determination of the abundance of ¹³C-¹³C bonds (i.e., ¹³Csubstitutions in two adjacent sites of the same molecule), anddetermination of the abundance of ¹³C-D bonds in methyl groups from theterminal positions of chain alkane structures (i.e., ¹³C substitutionsimmediately adjacent to a D substitution for H in the same methylgroup).

Determination of the ¹³C/¹²C ratios of non-equivalent sites in alkanestructures can be accomplished by determining the ¹³C/¹²C ratios ofintact molecular analyte ions and of analyte fragment ions, andcombining these data using mathematical expressions such as thosedescribed above (e.g., statistical determinations of isotopedistributions). This approach is applicable to n-alkanes containing 3 ormore carbons (i.e., propane and higher order hydrocarbons), andgenerally requires determination of one independent carbon isotope ratio(i.e., the ¹³C/¹²C ratio of one analyte molecule or molecular analytefragment) per non-equivalent carbon site.

Determination of the D/H ratios of non-equivalent sites in alkanestructures can be accomplished by determining the D/H ratios of intactmolecular analyte ions and of analyte fragment ions, and combining thesedata using mathematical expressions similar to those described above(e.g., statistical determinations of isotope distributions). Thisapproach is applicable to n-alkanes containing 3 or more carbons (i.e.,propane and higher order hydrocarbons), and generally requiresdetermination of one independent hydrogen isotope ratio (i.e., the D/Hratio of one analyte molecule or molecular analyte fragment) pernon-equivalent carbon site. Such measurements likely will requirecalibration of K_(migration) analytical constants for some compounds andanalytical conditions.

Determination of the abundance of ¹³C-¹³C bonds (i.e., ¹³C substitutionsin two adjacent sites of the same molecule) can be accomplished throughprinciples similar to those described above (e.g., statisticaldeterminations of isotope distributions), but can be constrained bymeasurements of abundances of doubly-substituted molecular analyte ions(e.g., ¹³C₂ ¹²CH₈ propane) and their analyte fragments (e.g., ¹³C₂H₅derived from the fragmentation of propane). Such measurements arepossible for any species containing two or more adjacent carbon atoms(i.e., ethane and larger n-alkanes).

Determination of the abundance of ¹³C-D bonds in methyl groups from theterminal positions of chain alkane structures (i.e., ¹³C substitutionsimmediately adjacent to a D substitution for H in the same methyl group)can be accomplished through analysis of the ¹³CH₂D/¹²CH₃ ratio ofanalyte methyl fragment ions (a minor but common species generated byelectron impact ionization of alkanes).

The largest molecular analyte or analyte fragment ion mass that can besubjected to these measurements will vary as a function of analyticalconditions (e.g., source water pressure, total pressure and tuningconditions), the level of precision desired, the strategy employed inion collection (e.g., multi-collection or peak scanning), and the methodof data processing (e.g., ion correction of non-resolved isobaricinterferences). In some instances, the isotopic composition of the fullanalyte molecule can be easily and precisely determined using previouslyexisting methods, and additional constraints from measurements ofanalyte fragment ions can be added according to embodiments of thepresent invention. Table 3 shows anticipated signal strengths formultiply substituted alkanes for a modest source pressure and anentrance slit having a width of about 5 μm.

TABLE 3 Isotopes abundance count rate time to 0.3% 1 × ¹³C 6 · 10⁻² 3 pA 1 s 2 × ¹³C 4 · 10⁻³ 1 · 10⁶ cps  9 s 3 × ¹³C 2 · 10⁻⁴ 5 · 10⁴ cps 200s 1 × ¹³C; 1 × D 1 · 10⁻⁴ 4 · 10⁴ cps 230 s

Embodiments of the present invention provide an increased number ofcompositional dimensions that can be investigated, which provides theopportunity to dramatically improve the specificity of “fingerprinting.” For example, conventional analysis of n-alkanes yields twoisotope ratios: ¹³C/¹²C and D/H. However, a comprehensive analysisaccording to embodiments of the present invention would yield: 10 ratiosfor methane, 128 ratios for ethane, 512 ratios for propane, ˜4,000ratios for n-butane, ˜32,000 ratios for n-pentane, and ˜10⁸ ratios forn-octane.

Example 3: Isotopic Anatomy of Glucose

Yet another embodiment of the invention is directed to the analysis ofthe proportions of ¹³C, D and/or ¹⁸O bearing isotopologues of ionfragments generated by delivering volatile organic compounds, such asderivatized sugars, into the ion source. The foregoing data, combinedwith characterizations of the empirical constants describingfragmentation and recombination reactions in the ion source, will allowfor the characterization of isotopic fingerprints associated withdiverse sources of such compounds and thus aid in the forensic studiesof diverse organic compounds (functionally, any species that can bederivatized to create a compound that can be delivered to the ion sourcethrough a heated gas chromatographic column).

For example, a method for the diagnosis or treatment of a diseaseincludes analyzing an analyte of a sample from a patient using anembodiment of the second mass spectrometer and/or methods describedherein to obtain the isotopic composition of at least a portion of theanalyte. The method also includes comparing the isotopic compositionobtained for the analyte to a database of isotopic compositions. Thecorrelation between the isotopic composition obtained for the analyteand the database of the isotopic compositions can be used to determine adisease diagnosis or disease treatment protocol.

In another embodiment of the invention, a method of analyzing a drug ordrug metabolite includes analyzing the drug or drug metabolite in asample using an embodiment of the second mass spectrometer and/ormethods described herein to obtain the isotopic composition of at leasta portion of the drug or drug metabolite. The method further includescomparing the isotopic composition obtained for the drug or drugmetabolite to a database of isotopic compositions. The correlationbetween the isotopic composition obtained for the drug or drugmetabolite and the database of isotopic compositions can be used todetermine a property of the drug or the drug metabolite and is useful inthe forensic study of diverse organic compounds.

Metabolic consumption of glucose in living organisms is characterized byisotopic fractionation of the residual glucose pool (e.g., change inisotopic composition of blood glucose as a function of the fractionconsumed). Though many of the details of this fractionation are unknown,the principles of chemical physics relevant to chemical separation ofisotopes indicate these effects can differ significantly with theconditions of glucose consumption (e.g., temperature) and the mechanismof consumption, possibly including subtle variations in the structuresof relevant enzymes and other reactive sites. Thus, characterization ofthe isotopic anatomy of glucose could serve as a diagnostic tool forcharacterizing the function of metabolic processes relevant to diabetesand possibly other diseases. Accordingly, aspects of embodiments of theinvention are directed toward determining the isotopic signatures ofmetabolites, including but not limited to the carbon, oxygen andhydrogen isotope compositions of components of the glucose molecule.Similar principles apply to other examples of biomolecules that aresubject to metabolic consumption.

Glucose and its derivatives can be transmitted through a gaschromatograph and thus are suitable analytes for embodiments of thepresent invention. Glucose itself has slow transport times throughconventional gas chromatograph columns, suggesting that, in someembodiments, the analysis might be better made on a faster-movingglucose derivative (e.g., a glucose derivative that elutes from a gaschromatograph column faster than glucose). On the other hand, componentsadded to glucose derivatives can contribute substantially to someportions of the mass spectrum, which can complicate interpretation ofthe isotopic measurements of glucose derivatives. Nonetheless, those ofordinary skill in the art can properly select glucose derivatives thatprovide suitable measurement characteristics. Regardless, both options(direct analysis of glucose or analysis of a glucose derivative) yieldanalyzable products on electron impact ionization and so are suitablefor analysis according to embodiments of the present invention.

The fragmentation spectrum of glucose under electron impact ionizationincludes more than 75 analyzable peaks in a conventional, low-resolutionmass spectrum; each of these peaks is the result of several analyte ionbeams corresponding to species that have the same cardinal mass (e.g.,¹³C and ¹²CH, at mass 13 AMU, etc.). Many of these composite peaks occurbelow mass 60 AMU, and thus should contain one or more component analyteion beams that can be uniquely mass resolved by the mass spectrometricanalyzer according to embodiments of the invention, assuming a massresolution of 20,000 or more. Others of these ion beams may be moredifficult to mass resolve, but are possibly analyzable using methodssuch as peak scanning and ion correction of non-resolved isobaricinterferences, as described above.

Take for example, the analyte ion beams of the glucose mass spectrumincluding analyte ions having respective masses of 28, 29 and 30 AMU;the corresponding portions of the glucose mass spectrum include largecontributions from CO and its hydrogen adducts (e.g., ¹²C¹⁶O at mass 28AMU, ¹²CO¹⁶H, ¹³C¹⁶O and ¹²C¹⁷O at 29 AMU, and ¹²C¹⁶OH₂, ¹³C¹⁶OH,¹²C₁₆OD, ¹²C¹⁸O and ¹³C¹⁷O at mass 30). The hydrogen bearing species ofCO are easily mass resolvable from the non-hydrogen bearing species,whereas isobaric interferences among the isotopologues of the COmolecule are not easily mass resolvable. Thus, in some embodiments, onecan measure the ratios: ([¹³C¹⁶O]+¹²C¹⁷O])/[¹²C¹⁶O] and(¹²C¹⁸O+¹³C¹⁷O])/[¹²C¹⁶O], as all common terrestrial materials share acommon relationship between ¹⁷O/¹⁶O(R¹⁷) and ¹⁸O/¹⁶O (R¹⁸): (R¹⁷_(sample)/R¹⁷ _(seawater))=(R¹⁸ _(sample)/R¹⁸ _(seawater))^(0.528).

Combination of the two measured ratios listed above (e.g.([¹³C¹⁶O]+¹²C¹⁷O])/[¹²C¹⁶O], and (¹²C¹⁸O+¹³C¹⁷O])/[¹²C¹⁶O]) withconstraints on R¹⁷ and R¹⁸ permits unique solution for the ¹³C/¹²C ratioand ¹⁸O/¹⁶O ratio of the CO fragment of glucose. This fragment isderived from the C═O double bond group at the terminal (C1) position ofthe glucose molecule. Possible contributions from fragments andrecombination products of other carbon and oxygen positions in themolecule are possible; the proportions of these contributions can bedetermined through analysis of synthetic isotopically labeled standards(this is effectively a case where standardization can be achievedthrough constraints on both K_(migration) and K_(redistribution)). Thisexample illustrates two pieces of information (e.g., ¹³C and ¹⁸O contentof CO) that can be obtained from one of the known fragment ion peaks ofglucose, and illustrates just a small fraction of all of the analyzablespecies.

According to another embodiment of the invention, a method fordetermining a prior temperature of a sample includes analyzing ananalyte of the sample using an embodiment of the second massspectrometer described herein to obtain molecular analyte ion data. Themethod further includes analyzing the molecular analyte ion data toobtain the isotopic composition of at least a portion of the analyte.For example, the method can include determining the isotopic compositionof at least a portion of an analyte in the sample according to one ofthe methods described herein. The method also includes comparing theisotopic composition obtained for the analyte to a database of isotopiccompositions. The method further includes determining the priortemperature of the sample based on the correlation between the isotopiccomposition obtained for the analyte and the database of isotopiccompositions.

According to another embodiment of the invention, a method ofdetermining an amount of an anthropogenic contribution to an atmosphericconcentration of an atmospheric gas includes analyzing an analyte of asample using an embodiment of the second mass spectrometer describedherein to obtain molecular analyte ion data. The method further includesanalyzing the molecular analyte ion data to obtain the isotopiccomposition of at least a portion of the analyte. For example, themethod can include determining the isotopic composition of at least aportion of an analyte in the sample according to one of the methodsdescribed herein. The method further includes comparing the isotopiccomposition obtained for the analyte to a database of isotopiccompositions. The method also includes determining the amount of theanthropogenic contribution to the atmospheric concentration of theatmospheric gas based on the correlation between the isotopiccomposition obtained for the analyte and the database of the isotopiccompositions. In some embodiments, the analyte is one or more ofmethane, carbon dioxide, sulfates, hydrocarbons, noble gases, and simplevolatile molecular species such as H₂, O₂, N₂, NO, and N₂O.

While the present invention has been described in connection withcertain exemplary embodiments, it is to be understood that the inventionis not limited to the disclosed embodiments, but, on the contrary, isintended to cover various modifications and equivalent arrangementsincluded within the spirit and scope of the appended claims, andequivalents thereof. Throughout the text and claims, use of the word“about” reflects the penumbra of variation associated with measurement,significant figures, and interchangeability, all as understood by aperson having ordinary skill in the art to which this inventionpertains. Additionally, throughout this disclosure and the accompanyingclaims, it is understood that even those ranges that may not use theterm “about” to describe the high and low values are also implicitlymodified by that term, unless otherwise specified.

What is claimed is:
 1. A mass spectrometer, the mass spectrometercomprising: a first ion travel path; a reference reservoir containing areference material comprising a known ratio of isotopologues; a firstintroduction device comprising one or more valves configured toselectively cycle between the reference material and a first portion ofan analyte and introduce the selected one of the reference material orthe first portion of the analyte to a first ion source, the first ionsource having a first entrance slit, the first entrance slit having afirst width, the first ion source configured to convert the referencematerial or the first portion of the analyte to first molecular analyteions and to guide the first molecular analyte ions along the first iontravel path, each of the first molecular analyte ions having a momentum;a first momentum filter positioned downstream from the first ion sourceand configured to receive the first molecular analyte ions, the firstmomentum filter having a first radius of curvature along the first iontravel path, the first momentum filter configured to filter out secondmolecular analyte ions from the first molecular analyte ions accordingto their momenta, each of the second molecular analyte ions having anenergy level; a first energy filter positioned downstream from the firstmomentum filter and configured to receive the second molecular analyteions, the first energy filter having a second radius of curvature alongthe first ion travel path, the first energy filter configured to producea double focusing condition at detector positioned downstream of thefirst energy filter, the detector configured to receive the secondmolecular analyte ions, wherein the width of the first entrance slit andthe first and second radii of curvature are selected to provide a firstmass resolution at the detector of about 30,000 or greater.
 2. The massspectrometer of claim 1, wherein the detector comprises a singlecollector, and the single collector is configured to detect the secondmolecular analyte ions.
 3. The mass spectrometer of claim 1, whereineach of the second molecular analyte ions has a mass that differs fromthe masses of the other of the second molecular analyte ions by lessthan 1 atomic mass unit.
 4. The mass spectrometer of claim 1, whereinthe first introduction device is configured to receive the first portionof the analyte as a gas phase analyte.
 5. The mass spectrometer of claim1, wherein: the analyte is a gas phase analyte and the firstintroduction device comprises a first inlet coupled to a samplereservoir configured to accommodate the gas phase analyte, the referencematerial is a gas phase reference material and the first introductiondevice comprises a second inlet coupled to a reference reservoirconfigured to accommodate the gas phase reference material, the samplereservoir is configured to accommodate the gas phase analyte at a firstpressure, the reference reservoir is configured to accommodate the gasphase reference material at a second pressure, and the first and secondpressures are the same.
 6. The mass spectrometer of claim 1, wherein thefirst introduction device is configured to receive the first portion ofthe analyte entrained in a flow of inert gas.
 7. The mass spectrometerof claim 1, wherein the first momentum filter is configured to produce amagnetic field, the first energy filter is configured to produce anelectric field, and the mass spectrometer is configured to maintain astrength of the magnetic field of the first momentum filter at a setvalue and to vary a strength of the electric field of the first energyfilter such that masses of the second molecular analyte ions detected atthe detector are varied.
 8. The mass spectrometer of claim 7, whereinthe second molecular analyte ions detected at the detector have the samecardinal mass.
 9. The mass spectrometer of claim 7, wherein the massspectrometer is configured to vary the strength of the electric field ofthe energy filter in a set range to vary the masses of the secondmolecular analyte ions detected at the detector in a range spanning oneatomic mass unit.
 10. The mass spectrometer of claim 9, wherein the massspectrometer is configured to vary the strength of the electric field ofthe first energy filter across the set range a plurality of times toproduce a plurality of spectra corresponding to the range spanning oneatomic mass unit.
 11. The mass spectrometer of claim 10, furthercomprising a processor configured to produce a model of each of thespectra, and to average the models to produce a modeled spectrum. 12.The mass spectrometer of claim 10, further comprising a processorconfigured to average the plurality of spectra to produce an averagedspectrum and to produce a model of the averaged spectrum.
 13. The massspectrometer of claim 1, wherein: the first momentum filter isconfigured to produce a magnetic field, the first energy filter isconfigured to produce an electric field, the mass spectrometer isconfigured to maintain a first strength of the magnetic field of thefirst momentum filter at a first set value and to vary a strength of theelectric field of the first energy filter such that first masses of thesecond molecular analyte ions detected at the detector are varied, andthe mass spectrometer is configured to maintain a second strength of themagnetic field of the first momentum filter at a second set value and tovary a strength of the electric field of the first energy filter suchthat second masses of the second molecular analyte ions detected at thedetector are varied.
 14. The mass spectrometer of claim 13, wherein: themass spectrometer is configured to vary the first strength of theelectric field of the first energy filter in a first set range such thatthe first masses of the second molecular analyte ions detected at thedetector are varied in a first range spanning one atomic mass unit, andthe mass spectrometer is configured to vary the second strength of theelectric field of the first energy filter in a second set range suchthat the second masses of the second molecular analyte ions detected atthe detector are varied in a second range spanning one atomic mass unit.15. A system for analyzing an analyte, the system comprising: a firstmass spectrometer comprising the mass spectrometer of claim 1; and asecond mass spectrometer comprising: a second ion travel path; a secondion source having a second entrance slit, the second entrance slithaving a second width, the second ion source being configured to converta second portion of the analyte to third molecular analyte ions and toguide the third molecular analyte ions along the second ion travel path,each of the third molecular analyte ions having an energy level; asecond energy filter positioned downstream from the second ion sourceand configured to receive the third molecular analyte ions, the secondenergy filter having a third radius of curvature along the second iontravel path, the second energy filter configured to produce a doublefocusing condition at a second detector, each of the third molecularanalyte ions having a momentum; and a second momentum filter positioneddownstream from the second energy filter and configured to receive thethird molecular analyte ions, the second momentum filter having a fourthradius of curvature along the second ion travel path, the secondmomentum filter configured to filter out fourth molecular analyte ionsfrom the third molecular analyte ions according to their momenta,wherein the second detector comprises a detector array positioneddownstream of the second momentum filter and configured to receive thefourth molecular analyte ions, wherein the second width, and third andfourth radii of curvature are selected to provide a second massresolution at the detector array of about 20,000 or greater.
 16. Thesystem of claim 15, further comprising a processor configured to processfirst molecular analyte ion data from the first mass spectrometer andsecond molecular analyte ion data from the second mass spectrometer. 17.The system of claim 16, wherein the processor comprises a firstprocessor configured to process the first molecular analyte ion data anda second processor configured to process the second molecular analyteion data.
 18. A method of identifying a potential oil-field, the methodcomprising: analyzing a sample from a target field using the system ofclaim 15 to obtain molecular analyte ion data, wherein the samplecomprises the analyte; analyzing the molecular analyte ion data toobtain an isotopic composition of at least a portion of the analyte todetermine relative proportions of at least a portion of isotopologues inthe sample; and comparing the relative proportions of the isotopologuesof the analyte to a database.
 19. A method of analyzing a drug or a drugmetabolite, the method comprising: analyzing a sample of the drug ordrug metabolite using the system of claim 15 to convert the drug or drugmetabolite to molecular analyte ions and to obtain molecular analyte iondata, wherein the sample comprises the analyte; analyzing the molecularanalyte ion data to obtain an isotopic composition of at least a portionof the drug or drug metabolite; and comparing the isotopic compositionobtained for the drug or drug metabolite to a database of isotopiccompositions.
 20. A method of determining an amount of an anthropogeniccontribution to an atmospheric concentration of an atmospheric gas, themethod comprising: analyzing a sample of the atmospheric gas using thesystem of claim 15 to obtain molecular analyte ion data, wherein thesample comprises the analyte; analyzing the molecular analyte ion datato obtain an isotopic composition of at least a portion of the analyte;and comparing the isotopic composition obtained for the analyte to adatabase of isotopic compositions.
 21. A method for diagnosing ortreating a disease, the method comprising: analyzing a sample from apatient using the system of claim 15 to obtain molecular analyte iondata, wherein the sample comprises the analyte; analyzing the molecularanalyte ion data to obtain an isotopic composition of at least a portionof the analyte; and comparing the isotopic composition obtained for theanalyte to a database of isotopic compositions.
 22. A method fordetermining a prior temperature of a sample, the method comprising:analyzing the sample using the system of claim 15 to obtain molecularanalyte ion data, wherein the sample comprises the analyte; analyzingthe molecular analyte ion data to obtain an isotopic composition of atleast a portion of the analyte; and comparing the isotopic compositionobtained for the analyte to a database of isotopic compositions.
 23. Themass spectrometer of claim 1, further comprising: a first conduitconfigured to provide the analyte from a first reservoir to the firstintroduction device; a second conduit configured to provide thereference material from the reference reservoir to the firstintroduction device; and a third conduit configured to provide at leastone of the analyte and the reference material to the first introductiondevice entrained in a flow of an inert carrier gas.
 24. A method fordetermining the isotopic composition of an analyte in a sample, themethod comprising: selectively cycling between a reference materialcomprising known isotopic compositions and a first portion of theanalyte with a first introduction device; producing an alternating flowof the reference material comprising known isotopic compositions and thefirst portion of the analyte to a first ion source; converting a firstportion of the analyte to first molecular analyte ions using a first ionsource of a first mass spectrometer; filtering out second molecularanalyte ions from the first molecular analyte ions according to theirmomenta; creating a double focusing condition by passing the secondmolecular analyte ions to a detector according to their energy levels;detecting two or more of the second molecular analyte ions at a massresolution of about 30,000 or greater to produce first molecular analyteion data; and analyzing the first molecular analyte ion data todetermine an isotopic composition of at least a portion of the analyteand to determine a molecular position from among a plurality ofmolecular positions of at least one isotope in the analyte.
 25. Themethod of claim 24, wherein the two or more of the second molecularanalyte ions have respective masses that are the same when rounded tothe nearest whole number, and the first molecular analyte ion datacomprises a separate, mass resolved signal for each of the two or moreof the second molecular analyte ions.
 26. The method of claim 24,wherein the second molecular analyte ions comprise two or more firstmolecular analyte ion beams, and the detecting the two or more of thesecond molecular analyte ions comprises scanning at least two of thefirst molecular analyte ion beams across a single detector.
 27. Themethod of claim 26, wherein the second molecular analyte ions of each ofthe two or more first molecular analyte ion beams have masses thatdiffer from one another by less than about 1 atomic mass unit.
 28. Themethod of claim 24, further comprising introducing the first portion ofthe analyte to the first mass spectrometer as a continuous flow prior toconverting the first portion of the analyte to the first molecularanalyte ions.
 29. The method of claim 24, further comprising introducingthe first portion of the analyte to the first mass spectrometer as atime-resolved pulse prior to converting the first portion of the analyteto the first molecular analyte ions.
 30. The method of claim 24, whereinthe analyte comprises two or more analyte isotopologues, analyteisotopomers or mixtures thereof.
 31. The method of claim 30, wherein theanalyzing further comprises determining the molecular position of the atleast one isotope in at least one of the analyte isotopologues or theanalyte isotopomers.
 32. The method of claim 24, further comprising:converting a second portion of the analyte to third molecular analyteions using a second ion source in a second mass spectrometer; creating asecond double focusing condition at a detector in the second massspectrometer by passing the third molecular analyte ions according totheir energy levels; filtering out fourth molecular analyte ions fromthe third molecular analyte ions according to their momenta; detectingtwo or more of the fourth molecular analyte ions at a second massresolution of about 20,000 or greater to produce second molecularanalyte ion data; and analyzing the second molecular analyte ion data todetermine an isotopic composition of at least a portion of the analyte.33. A method of identifying a potential oil-field, the methodcomprising: determining an isotopic composition of a sample from atarget field according to the method of claim 24 to determine relativeproportions of at least a portion of isotopologues in the sample,wherein the sample comprises the analyte; comparing the relativeproportions of the isotopologues of the analyte to a database.
 34. Amethod of analyzing a drug or a drug metabolite, the method comprising:determining an isotopic composition of a sample of the drug or the drugmetabolite according to the method of claim 24, wherein the samplecomprises the analyte; and comparing an isotopic composition obtainedfor the drug or drug metabolite to a database of isotopic compositions.35. A method of determining an amount of an anthropogenic contributionto an atmospheric concentration of an atmospheric gas, the methodcomprising: determining an isotopic composition of a sample of theatmospheric gas according to the method of claim 24, wherein the samplecomprises the analyte; and comparing the isotopic composition obtainedfor the analyte to a database of isotopic compositions.
 36. A method fordiagnosing or treating a disease, the method comprising: determining anisotopic composition of a sample from a patient according to the methodof claim 24, wherein the sample comprises the analyte; and comparing theisotopic composition obtained for the analyte to a database of isotopiccompositions.
 37. A method for determining a prior temperature of asample, the method comprising: determining an isotopic composition ofthe sample according to the method of claim 24, wherein the samplecomprises the analyte; and comparing the isotopic composition obtainedfor the analyte to a database of isotopic compositions.
 38. A massspectrometer, the mass spectrometer comprising: a first ion travel path;a reference reservoir containing a reference material comprising a knownratio of isotopologues; a first introduction device comprising one ormore valves configured to selectively cycle between the referencematerial and a first portion of an analyte and introduce the selectedone of the reference material or the first portion of the analyte to afirst ion source, the first ion source having a first entrance slit, thefirst entrance slit having a first width, the first ion sourceconfigured to convert the reference material or the first portion of theanalyte to first molecular analyte ions and to guide the first molecularanalyte ions along the first ion travel path, each of the firstmolecular analyte ions having a momentum; a first momentum filterpositioned downstream from the first ion source and configured toreceive the first molecular analyte ions, the first momentum filterhaving a first radius of curvature along the first ion travel path, thefirst momentum filter configured to filter out second molecular analyteions from the first molecular analyte ions according to their momenta,each of the second molecular analyte ions having an energy level; afirst energy filter positioned downstream from the first momentum filterand configured to receive the second molecular analyte ions, the firstenergy filter having a second radius of curvature along the first iontravel path, the first energy filter configured to produce a doublefocusing condition at a detector positioned downstream of the firstenergy filter, the detector comprising a single collector configured todetect the second molecular analyte ions, wherein the width of the firstentrance slit and the first and second radii of curvature are selectedto provide a first mass resolution at the detector of about 30,000 orgreater, wherein each of the second molecular analyte ions has a massthat differs from the masses of the other of the second molecularanalyte ions by less than 1 atomic mass unit, wherein the introductiondevice is configured to receive the first portion of the analyteentrained in a flow of inert gas or as a gas phase analyte, and whereinthe first momentum filter is configured to produce a magnetic field, thefirst energy filter is configured to produce an electric field, and themass spectrometer is configured to maintain a strength of the magneticfield of the first momentum filter at a set value and vary a strength ofthe electric field of the first energy filter in a set range to vary themasses of the second molecular analyte ions detected at the detector ina range spanning one atomic mass unit.